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Determination of an oral aflatoxin dose that acutely impairs hepatic function in domestic pigeons (Columba livia).

Abstract: Aflatoxin Bj is a common hepatotoxin in birds. The goal of this study was to establish an acute model for hepatotoxicosis and decreased hepatic function in the white Carneaux pigeon (Columba livia) via oral administration of this mycotoxin. Aflatoxin [B.sub.1] was orally administered at a dose of 3 mg/kg dissolved in dimethyl sulfoxide to 3 groups of pigeons every 24 hours for 2, 4, and 6 consecutive days, respectively. Diagnostic modalities used to evaluate hepatic damage and impaired hepatic function pre- and postaflatoxin administration included liver enzyme activity, bile acid levels, scintigraphy, and histopathologic evaluation of liver biopsy specimens. Deaths occurred in all groups, increasing with the number of consecutive days the aflatoxin BI was dosed. Significant histopathologic lesions were seen on evaluation of hepatic tissue from each group after accumulated aflatoxin exposure (P < .05); therefore, an oral aflatoxin BI dose of 3 mg/kg given for 2 consecutive days was selected for the purpose of inducing acute hepatic damage while minimizing mortality. However, although increased liver enzyme activity indicated hepatocellular damage at this dosage, bile acids testing and hepatobiliary scintigraphy did not show significantly decreased hepatic function.

Key words: aflatoxin [B.sub.1], mycotoxin, scintigraphy, 99mTc-mebrofenin, liver, bile acids, avian, pigeon, Columba livia

Introduction

Aflatoxin [B.sub.1] is one of the most common mycotoxins that affect birds. (1) Aflatoxicosis occurs as a result of the activity of secondary toxic metabolites produced by certain molds, particularly Aspergillus fumigatus and Aspergillus parasiticus. (23) These molds may be found on various food items, such as milk, meats, cheeses, and grains. (1,4) Aflatoxins may also occur naturally in peanuts and peanut meal, tree nuts (eg, coconut, almonds, pistachio, and walnuts), oilseeds (eg, soybean and sunflower), corn, and spices, such as black pepper and dried chili peppers. (3,4) The presence of mold on a food item does not always guarantee the presence of the toxin. (3) Certain environmental conditions (eg, temperature, moisture, aeration, and substrate) must be favorable for toxin formation. (3) Among all the demonstrated mycotoxins, aflatoxin [B.sub.1] is one of the most potent carcinogens and environmental mutagens. (3)

Aflatoxins may pose a threat to wildlife as well as captive avian species. (5-8) Results of research performed in chickens have shown that aflatoxins can cause toxic injury to the liver, with resultant lesions, including fatty degeneration, bile duct hyperplasia, and massive necrosis. (9,10) The kidneys may also be affected, which results in tubular degeneration. (11) Clinical signs of aflatoxicosis vary, depending on the degree and length of exposure; these include listlessness, ataxia, decreased activity, decreased feed consumption, and weight loss. (9) Birds may also experience gastrointestinal hemorrhage, an alteration in anticoagulant activity, and immunosuppression. (1) Currently, there is no known treatment for aflatoxicosis in birds.

Numerous studies have been undertaken in poultry to assess the effects of aflatoxin [B.sub.1] on performance and other parameters. In 1 study, in broiler chicks, aflatoxin [B.sub.1] caused decreased feed intake and decreased body weight gain. The same study also determined that the toxin increased alkaline phosphatase (ALP) serum activity, decreased serum protein concentration, and increased the relative weight of the liver. (12) Another study, in male broiler chicks, determined that aflatoxin [B.sub.1] at a concentration of 2 mg/kg resulted in altered gene expression in chick livers and secondary changes in physiological responses. (13) Studies on production performance in female white Leghorn chicks showed a delay in sexual maturity in birds fed relatively higher doses of aflatoxin [B.sub.1]. (14) Adverse histologic changes were also seen in the bursa of Fabricius and in ovaries. Exposure to aflatoxins may also induce depletion of hepatic vitamin A stores, result in depressed hepatic blood perfusion in chicks, and lead to a decreased antibody response. (15-17)

Compared with broiler chicks, turkey poults are considered 3-6 times more sensitive to the effects of aflatoxin poisoning. A study that evaluated the effects of aflatoxins on turkey poults showed both increases and decreases in the plasma protein and cholesterol levels at various aflatoxin concentrations. (18) The high sensitivity of domestic turkeys to aflatoxin exposure has also brought attention to the toxin's possible effects on wild turkeys that take part in public feeding or baiting practices. (19) Wild turkey poults (Cenus species) fed various doses of aflatoxins showed decreased feed intake and weight gain as well as changes in liver enzyme activities. Japanese quail (Coturnix japonicus) are also affected by aflatoxin exposure. In 1 study, vacuolation and fatty infiltration of hepatic cells was found in all quail fed low levels of aflatoxin [B.sub.1]. (20) Quail fed higher aflatoxin levels showed evidence of bile-duct proliferation.

The ability to assess the effect of aflatoxin [B.sub.1] exposure on the liver is based upon the evaluation of several clinicopathologic tests. Aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) enzyme activities are both commonly used to assess damage to hepatocytes. Although many tissues exhibit high AST activities, elevations in this enzyme typically occur because of changes in muscle or liver. Creatine phosphokinase (CK) enzyme activities, which usually result from changes in muscle, may be used to distinguish the causes of high AST activities. (21) CK values tend be elevated for a relatively short time after muscle necrosis. (22) Like AST, LDH activities occur in several avian tissues. However, increases in LDH enzyme activity are most commonly attributed to changes in the liver. Because LDH activities tend to rise and fall more rapidly than AST enzyme activities, evaluation of each of these values from a blood sample obtained during any point in time may suggest that the damage to the liver is either an acute or chronic phenomenon. (21)

Gamma glutamyltransferase (GGT) enzyme activities may also be used to assess specific changes to the liver. Increases in GGT are most commonly a result of injury to the biliary tree from inflammation, neoplasia, or obstruction (eg, from choleliths). (22) The bile acid assay is used to evaluate liver function; an increase in the bile acid level above normal generally indicates impaired hepatic function. (22) Other modalities used to evaluate the liver include radiography, endoscopy, hepatic cytology, culture, and biopsy. Hepatobiliary scintigraphy, a more recently developed modality, is being investigated to determine its sensitivity in detecting liver function changes. (23)

Materials and Methods

Birds

Twenty-one white Carneaux pigeons (Columba livia) were purchased from a commercial supplier (Palmetto Pigeon Plant, Sumter, SC, USA). The pigeons were determined to be healthy based upon results of physical examination; they weighed between 0.54 and 0.72 kg. The pigeons were housed in an environmentally controlled facility with an ambient temperature of 18[degrees]C21[degrees]C (65[degrees]F 70[degrees]F) and were kept on a 12-hour light cycle. They were caged individually or in groups of 2 in grower battery cages (Petersime Company, Gettysburgh, OH, USA) that measured 0.6 m x 0.6 m and were fed a commercial pelleted pigeon ration. The birds were acclimated to their environment for at least 2 weeks before the beginning of the study. During the aflatoxin portion of the study, the pigeons were housed in a biocontainment facility. The study protocol was approved by and in accordance with the guidelines of The University of Tennessee Institutional Animal Care and Use Committee.

Hepatic study in normal pigeons before aflatoxin exposure

This part of the study was performed according to previously described protocols. (23) The pigeons were fasted for approximately 12 hours before induction of anesthesia with 2% isoflurane delivered in oxygen via face mask. Each bird was intubated with a 2-mm uncuffed endotracheal tube, and anesthesia was maintained with 1.5%2.5% isoflurane in 100% oxygen. Blood samples were obtained from each bird and submitted for a complete blood count (CBC), plasma biochemical profile, and bile acid assay. The birds were positioned in dorsal recumbency on a transparent section of acrylic sheeting that measured 46 cm x 25 cm, with a thickness of 0.63 cm. The acrylic board was used to mimic the boxes that would house the aflatoxin-exposed pigeons so as to provide consistency of values obtained during hepatobiliary scintigraphy.

The acrylic board that held the anesthetized pigeons was placed on a table over a large field-of-view gamma camera (GE 400 Gamma Camera, Fairfield, CT, USA) fitted with a low-energy, all-purpose parallel hole collimator and interfaced with a dedicated imaging computer (NucLear Mac, Scientific Imaging Inc, Littleton, CO, USA). The birds were held in position with masking tape. A 26-gauge over-the-needle intravenous catheter (Becton Dickinson Infusion Therapy Systems Inc, Sandy, UT, USA) was placed in either the left or right basilic vein and was stabilized to the skin with a single drop of tissue glue (Nexaband, Abbott Laboratories, Abbott Park, IL, USA). The intravenous catheter was flushed with 0.2 mL heparinized saline solution (2 IU heparin/mL).

A radioconjugate of mebrofenin bound to an isotope of technetium, Tc-mebrofenin, was used for scintigraphy. The mebrofenin kit (Choletec, Bracci Diagnostics, Princeton, NJ, USA) was reconstituted according to the manufacturer's recommendations. The maximum recommended quantity of pertechnetate (3700 MBq, or 100 mCi, in 6 mL of sterile eluate) was used to reconstitute the kit to minimize the volume of mebrofenin to be injected. Radiochemical purity was assessed by using 2 ascending chromatographic systems: a silica gel-impregnated instant thin-layer chromatography paper developed with distilled water and a silica acid-type instant thin-layer chromatography paper developed with 20% sodium chloride.

A bolus injection of 55.5-74 MBq (1.5-2.0 mCi, 1.0 mL total volume) 99mTc-mebrofenin was administered by the intravenous catheter, which was immediately followed by 0.2 mL heparinized saline solution. Scintigraphic imaging was started immediately before injection of the radiopharmaceutical. A multiphase dynamic frame-mode acquisition was obtained at a frame rate of 1 frame per 5 seconds for the first 5 minutes then 1 frame per 30 seconds for an additional 35 minutes for a total acquisition time of 40 minutes. The images were stored in a 128 x 128 x 16 matrix.

At the completion of each scintigraphy period, hepatic biopsy samples were obtained from each pigeon and submitted for histopathologic examination. Butorphanol (2.0 mg/kg IM; Torbugesic, Fort Dodge Animal Health, Wyeth, Madison, N J, USA) was administered for analgesia, and the birds were placed in dorsal recumbency. The ventral abdomen was aseptically prepared, and a small midline incision was made into the hepatic peritoneal cavity immediately ventral to the sternum. Endoscopic biopsy forceps or mosquito hemostats were used to obtain 2 hepatic specimens. Hemorrhage from the biopsy site was minimized by applying gentle pressure with cotton-tipped applicators. The skin incision and peritoneal cavity were closed in 1 layer by using 4-0 polydioxanone (PDS, Ethicon Inc, Somerville, NJ, USA) in a Ford interlocking pattern. Each liver specimen was evaluated histologically by 1 pathologist (D.S.R.) who was blinded to the identity of the pigeons. The specimens were evaluated in 4 categories based upon a previously described grading scale. (24,25) A smaller percentage or lower score indicated less hepatic damage, whereas a greater percentage or higher score indicated greater hepatic injury.

Hepatic study in aflatoxin-exposed pigeons

A dimethyl sulfoxide (DMSO) tolerance test was performed to assess potential negative effects. Five pigeons were gavaged with 0.4 mL of 99.7% pure DMSO (Sigma-Aldrich, St Louis, MO, USA) for 6 consecutive days by using a 1-mL tuberculin syringe (Bectin-Dickinson Company, Rutherford, NJ, USA). Their response to DMSO was evaluated based upon activity level, appetite, fecal production, and presence of regurgitation.

Twenty-one pigeons were randomly divided into 3 groups of 7 birds. Each bird was weighed 1 day before oral administration of aflatoxin BI. A lyophilized powder of aflatoxin [B.sub.1] (Sigma-Aldrich) was suspended in DMSO to a final concentration of 10 mg/mL. Group A birds were administered aflatoxin [B.sub.1] (3 mg/kg) orally once daily for 2 consecutive days, group B birds were administered aflatoxin [B.sub.1] (3 mg/kg) orally once daily for 4 consecutive days, and group C birds were administered aflatoxin [B.sub.1] (3 mg/kg) orally once daily for 6 consecutive days. All birds in group C died, so a group D was created by taking 1 pigeon originally assigned to group A and 3 pigeons originally assigned to group B; pigeons in group D received aflatoxin (3 mg/kg) orally once daily for 3 days.

The daily oral dose of 3 mg/kg of aflatoxin [B.sub.1] was based upon previous studies in which aflatoxin [B.sub.1] was used to induce hepatic disease in birds for evaluation of the sensitivity of selected biochemical tests. (26,27) The aflatoxin [B.sub.1] was gavaged by using a 1-mL tuberculin syringe. No bird received more than 0.3 mL of aflatoxin [B.sub.1] dissolved in DMSO. During the study, each bird was evaluated daily for pain and discomfort based upon decreased activity, anorexia, regurgitation, and incoordination. Birds were fasted for 2 hours on the evening of their final aflatoxin [B.sub.1] dose to minimize aspiration from the typically large amount of ingesta held in their crops.

The day after their final dose, the birds were individually placed in boxes constructed of the same transparent acrylic material that was used in the normal pigeon study detailed previously. The acrylic box measured 53 cm x 50 cm x 38 cm and served to eliminate human exposure to latent aflatoxin. Each bird was anesthetized in the acrylic box, and blood samples were obtained for a CBC, plasma biochemical analysis, and bile acid assay. Hepatobiliary scintigraphy was performed 2430 hours after the final dose of aflatoxin according to a previously described procedure. (23) After scintigraphy, the birds were euthanatized with an intravenous injection of 0.3 mL pentobarbital sodium. A section of liver approximately 3 cm wide was removed from each bird and submitted for histopathologic examination.

Scintigraphic data analysis

Regions of interest were drawn over a portion of the liver that did not overlap a major biliary duct. In addition, regions of interest were drawn over the heart. The heart radioactivity was used as a measure of the changing plasma activity. The count density (counts/pixel) from both regions was used to generate the liver and heart time-activity curves. Tabulated data from these curves were then imported into a spreadsheet program (Excel 2003, Microsoft, Redmond, WA, USA) and converted into data suitable for statistical analysis as previously described in the dog and pigeon. (23-25) The liver and heart time-activity curves were then imported into a mathematic program (IgorPro 3.2, Wavemetrics, Oswego, OR, USA) for deconvolutional analysis.

Scintigraphic parameters measured included megabecquerels of 99mTc, milligrams of mebrofenin, the area under the normalized heart time-activity curve, fast and slow phases of [T.sub.1/2] plasma clearance of the radionucleotide, hepatic excretion, hepatic extraction rate, and the time to maximum liver uptake. In pigeons, the area under the heart time-activity curve was considered the best measure of hepatic cell function. (23) All parameters were statistically evaluated and compared between the pre- and postaflatoxin [B.sub.1] groups by using a paired t test when the data were normally distributed and a Wilcoxon signed rank test for data failing normality testing. Pearson product movement correlations were used to examine the relationship between the postaflatoxin indices of liver function and the overall histologic grade of the liver samples taken at necropsy. The level of significance was set at P < .05.

Results

None of the 5 pigeons that received DMSO in the tolerance trial showed any abnormal clinical signs either during or after the trial. Deaths occurred in all aflatoxin [B.sub.1]-treated groups, excluding the newly created group D. Deaths included 1 pigeon in group A, 2 pigeons in group B, and all 7 pigeons in group C. Therefore, hepatobiliary scintigraphy was performed on 5 pigeons from group A, 2 pigeons from group B, and 4 pigeons from group D.

Clinical pathology results

Hematologic and biochemical mean values before and after exposure to aflatoxin are listed in Tables 1 and 2, respectively. Several significant differences were seen in the parameters measured after aflatoxin administration. Birds in group A showed a significant increase in AST activity (P < .05). In birds in group D, the lymphocyte count and cholesterol concentration were significantly decreased (P < .05), but significant increases were seen in the WBC count, LDH and AST activities, and bile acid level (P < .01), as well as the heterophil and monocyte counts and the phosphorus and uric acid levels (P < .05). In group B, the hematocrit, cholesterol, and total protein levels were significantly decreased (P < .05), but a significant increase was seen in the phosphorus level and AST activity (P < .01), as well as the monocyte count, uric acid level, and LDH activity (P < .05).

Seintigraphy results

Birds in groups D and B showed significant increases in the area under the heart time-activity curve, the slow and fast phases of [T.sub.1/2] plasma clearance, and the time to maximum liver uptake (P < .05) after aflatoxin administration (Table 3). No significant changes in any scintigraphic parameters subsequent to aflatoxin administration were seen in the birds from group A.

[FIGURE 1 OMITTED]

Evaluation of hepatic biopsy specimens

Two hepatic specimens were evaluated and graded from each pigeon before and after exposure to oral aflatoxin [B.sub.1]. Before aflatoxin [B.sub.1] exposure, mild inflammation was detected in most of the hepatic specimens (Fig 1). After administration of aflatoxin [B.sub.1], all specimens showed evidence of moderate to severe hepatic changes, including lipidosis, mixed inflammatory cell infiltration, hepatocellular necrosis, hepatocyte regeneration, and biliary hyperplasia (Figs 2-4). Exposure to orally administered ariatoxin [B.sub.1] produced varying degrees of changes in the liver, which resulted in overall hepatic scores from 4 (n = 2) to 8 (n = 1). The degeneration score was significant for all exposure groups after oral aflatoxin [B.sub.1] exposure (P < .01) (Table 4). A significant increase in the inflammation score was also observed in group A pigeons (P < .01), and the necrosis score was significant in groups A and D (P < .05). The total hepatic score for each group was significantly increased after oral aflatoxin [B.sub.1] exposure (P < .01).

Discussion

The objective of this study was to determine the minimum consecutive days of acute oral aflatoxin [B.sub.1] poisoning needed to induce decreased hepatic function and hepatotoxicosis in white Carneaux pigeons. Based on histologic results, aflatoxin [B.sub.1] administered orally over 2 consecutive days at a dosage of 3 mg/kg q24h induced hepatotoxicosis. However, the plasma biochemical analysis and scintigraphic results did not support decreased hepatic function at this dosage. The likelihood of inducing decreased hepatic function was greater at higher cumulative dosages of aflatoxin [B.sub.1], but more deaths occurred at the higher dosages. As a result of these deaths, no further consideration was given to inducing decreased hepatic function beyond a dosage of 3 mg/kg of aflatoxin [B.sub.1] administered for 2 consecutive days.

[FIGURE 2 OMITTED]

The fatalities observed were likely the result of the high cumulative dose of oral aflatoxin [B.sub.1] administered. The deaths, starting as early as 2 days after oral aflatoxin [B.sub.1] exposure, occurred earlier than anticipated. In a previous study, adult racing homing pigeons received the same dose of oral aflatoxin [B.sub.1] once daily until death. (26) However, most deaths in those birds occurred at 7-10 days after initial dosing. The difference in response to aflatoxin [B.sub.1] may have resulted from a difference between pigeon strains. In this study, creation of a new pigeon group midway in the study was not optimal, but, under the circumstances, it was necessary to achieve the objectives of the study. Another consideration was that the DMSO, which was used to dissolve the aflatoxin [B.sub.1], may have had detrimental effects on the pigeons' health, in particular, the hepatic tissue. DMSO is labeled for topical use during situations of acute swelling that result from trauma, and it is well absorbed after topical application. (28) In horses, after intravenous use, it is excreted predominantly through the kidneys, although some excretion via the biliary and respiratory tracts may occur. Hepatotoxicity and renal toxicity may occur in various animal species, depending on the dosage used. In chickens, the LDs0 for oral DMSO administration occurred at 12 g/kg. (29) We are aware of no published data regarding the [LD.sub.50] for orally administered DMSO in pigeons. The dose of 99.7% pure DMSO used in this study was extremely small. In addition, the 5 pigeons that received DMSO in the tolerance trial showed no clinical signs during or after the trial. Ideally, in this study, a DMSO control group would have been set up for each aflatoxin treatment group. Therefore, we cannot state conclusively that the hepatic injury and other changes revealed in this study were solely attributable to the effects of aflatoxin [B.sub.1].

[FIGURE 3 OMITTED]

The decreased lymphocyte counts among all study groups (including a significant decrease in counts in group 15)) was thought to be a relative change that resulted from the increased heterophil counts. Results of the biochemical analyses showed expected increases in several enzymatic activities. The significant increases in AST and LDH activities in the face of normal CK activities in groups D and B suggested primary hepatic, rather than muscle, damage. In group A, only the AST and not the LDH activity was significantly increased. This finding is unusual when considering that, in situations of hepatic disease, LDH activities tend to rise and fall much faster than AST activities. (21) In addition, the 48-hour time period from initial aflatoxin [B.sub.1] dosing to blood sampling was short. The lack of increased bile acid values in group A pigeons suggested either that the dose of oral aflatoxin [B.sub.1] was not sufficiently high to result in a change in hepatic function, a longer period was needed after the final aflatoxin dose to see a significant change in liver function, or the bile acid assay was not sufficiently sensitive to detect a change in function.

In 1 study of racing pigeons, drug-induced hepatic damage resulted in plasma bile acid values 5-10 times higher than the upper limit of the reference range. The study determined that plasma bile acids were a specific and sensitive indicator of liver disease. (30) A second study found that increased bile acid levels had a higher correlation with confirmed liver disease than other clinical enzymatic activities, such as AST and CK. (31) In our study, the number of birds in group A may have been too small to appreciate a change in the bile acid value in the face of postaflatoxin liver damage. Interestingly, only birds in group D showed a significant increase in the bile acid levels, whereas, those in group B, the pigeons that had the longest exposure to oral aflatoxin [B.sub.1], did not show a significant increase. However, the study populations of groups D and B were also very small.

[FIGURE 4 OMITTED]

Hepatobiliary scintigraphy did not detect a significant increase in the area under the heart time-activity curve for group A, which suggested that the 2-day oral dose of aflatoxin [B.sub.1] was not sufficiently high to induce a decrease in hepatic function, the test was not sufficiently sensitive to detect a change in function, the time frame for testing after the last dose was too short, or the size of the group was too small. However, significant increases were observed in the area under the heart time-activity curve for both groups D and B, which suggested that either hepatobiliary scintigraphy could be more sensitive than bile acid evaluation for the detection of decreased hepatic function in pigeons in general or for the early detection of worsening hepatic function.

Histologic evaluation of hepatic biopsy specimens was a reliable indicator of hepatotoxicosis among all aflatoxin [B.sub.1]-exposed groups; however, it did not provide information on liver function. Although the study attempted to induce decreased hepatic function in pigeons when using oral aflatoxin [B.sub.1], fatalities subsequent to high dosing made it impossible to use dosages that resulted in scintigraphically detectable changes in function. The study showed that a daily oral dose of 3 mg/kg of aflatoxin [B.sub.1] administered over 2 consecutive days induced moderate-to-severe toxic changes in most hepatic specimens. Future studies may warrant evaluation of a lower and more chronic dosage of oral aflatoxin [B.sub.1] sufficient to result in significantly decreased hepatic function detectable either via bile acid evaluation or hepatobiliary scintigraphy.

Acknowledgments: We thank the Morris Animal Foundation for financial support of this research. We also thank Roger Long of the East Tennessee Research and Education Center for his expertise in construction of the transparent acrylic boxes used in the study.

References

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Tarah L. Hadley, DVM, Dipl ABVP, Judith Grizzle, PhD, David S. Rotstein, DVM, MPVM, Dipl ACVP, Shannon Perrin, Lillian E. Gerhardt, LVT, James D. Beam, LVT, Arnold M. Saxton, PhD, Michael P. Jones, DVM, Dipl ABVP, and Gregory B. Daniel, DVM, MS, Dipl ACVR

From the Departments of Small Animal Clinical Sciences (Hadley, Gerhardt, Beam, Jones, Daniel), Animal Science (Grizzle, Perrin, Saxton), and Pathobiology (Rotstein), College of Veterinary Medicine, University of Tennessee, 2407 River Dr, Knoxville, TN 37996, USA. Present address: Atlanta Hospital for Birds and Exotics, 2274 Salem Rd, 106149, Conyers, GA 30013, USA (Hadley); Department of Small Animal Clinical Sciences, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Tech, Duck Pond Dr, Blacksburg, VA 24061, USA (Daniel); University of Tennessee Extension, 602 John Deere Dr, Maynardville, TN, 378073510, USA (Perrin); and US Department of Commerce, National Oceanic and Atmospheric Administration, 1305 East West Highway, Silver Spring, MD 20910, USA (Rotstein).
Table 1. Results of hematologic and plasma biochemical analysis
(mean [+ or /] SD) of white Carneaux pigeons before oral
administration of aflatoxin B, (3 mg/kg to be given once daily
for 2, 3, or 4 consecutive days [groups A, D, or B,
respectively]).

Parameter                        Group A (n = 5)

Hematocrit (%)                 50.2 [ + or -] 4.9
Red blood cells                 4.0 [ + or -] 0.45
 (x [10.sup.6]/[micro]L)
White blood cells (x          16.78 [ + or -] 10.67
 [10.sup.3]/[micro]L) (b)
  Heterophils (%)              58.2 [ + or -] 18.5
  Lymphocytes (%)              41.2 [ + or -] 18.5
  Monocytes (%)                 0.6 [ + or -] 0.5
Alanine aminotransferase                --
 (IU/L)
Amylase (IU/L)                  443 [ + or -] 111
Aspartate aminotransferase   196.25 [ + or -] 88.88
 (IU/L)
Bile acids ([micro]mol/L)      3.51 [ + or -] 1.64
Blood urea nitrogen (mg/dL)             --
Calcium (mg/dL)                 8.8 [ + or -] 0.3
Carbon dioxide (mmol/L)                  --
Cholesterol (mg/dL)           234.0 [ + or -] 23.3
Creatine phosphokinase        267.0 [ + or -] 64.9
 (IU/L)
Creatinine (mg/dL)                      --
Gamma glutamyltransferase       5.0 [ + or -] 0
 (mg/dL)
Glucose (mg/dL)               313.3 [ + or -] 12.6
Lactate dehydrogenase           453 [ + or -] 133
 (IU/L)
Lipase (IU/L)                 100.0 [ + or -] 39.9
Phosphorus (mg/dL)             2.95 [+ or -] 0.94
Potassium (mmol/L)                      --
Sodium (mmol/L)                         --
Total protein (g/dL)            4.1 [ + or -] 0.9
Triglycerides (mg/dL)          82.5 [ + or -] 22.2
Uric acid (mg/dL)              2.85 [ + or -] 1.31

Parameter                       Group D (n = 4)

Hematocrit (%)               51.25 [ + or -] 4.3
Red blood cells               3.92 [ + or -] 0.49
 (x [10.sup.6]/[micro]L)
White blood cells (x          10.8 [ + or -] 2.6
 [10.sup.3]/[micro]L) (b)
  Heterophils (%)             45.0 [ + or -] 30.6
  Lymphocytes (%)             54.3 [ + or -] 30.2
  Monocytes (%)                0.8 [ + or -] 0.95
Alanine aminotransferase      36.5 [ + or -] 17.6
 (IU/L)
Amylase (IU/L)                 375 [ + or -] 74
Aspartate aminotransferase   146.0 [ + or -] 47.1
 (IU/L)
Bile acids ([micro]mol/L)     4.49 [ + or -] 7.38
Blood urea nitrogen (mg/dL)    2.0 [ + or -] 0
Calcium (mg/dL)                8.4 [ + or -] 0.6
Carbon dioxide (mmol/L)        30.0 [ + or -] 0
Cholesterol (mg/dL)          283.0 [ + or -] 13.2
Creatine phosphokinase       346.8 [ + or -] 82.2
 (IU/L)
Creatinine (mg/dL)             0.1 [ + or -] 0
Gamma glutamyltransferase      5.0 [ + or -] 0
 (mg/dL)
Glucose (mg/dL)              329.0 [ + or -] 27.4
Lactate dehydrogenase          265 [ + or -] 64
 (IU/L)
Lipase (IU/L)                139.3 [ + or -] 116
Phosphorus (mg/dL)             3.1 [ + or -] 0.6
Potassium (mmol/L)            1.65 [ + or -] 0.07
Sodium (mmol/L)              134.0 [ + or -]  0
Total protein (g/dL)          3.65 [ + or -] 0.68
Triglycerides (mg/dL)         75.7 [ + or -] 7.5
Uric acid (mg/dL)              1.9 [ + or -] 1.0

                                    Group B           Reference
Parameter                            (n = 2)          range (a)

Hematocrit (%)                 56.5 [ + or -] 2.1     39.3-59.4
Red blood cells                3.97 [ + or -] 0.05     2.1-4.2
 (x [10.sup.6]/[micro]L)
White blood cells (x           7.45 [+ or -] 2.75       10-30
 [10.sup.3]/[micro]L) (b)
  Heterophils (%)              48.5 [ + or -] 12.0      15-50
  Lymphocytes (%)              51.0 [+ or -] 11.3       25-70
  Monocytes (%)                 0.5 [ + or -] 0.7        1-3
Alanine aminotransferase               --               19-48
 (IU/L)
Amylase (IU/L)                  367 [ + or -] 0           --
Aspartate aminotransferase    147.0 [+ or -] 39.5       45-123
 (IU/L)
Bile acids ([micro]mol/L)       2.7 [+ or -] 0.4        22-60
Blood urea nitrogen (mg/dL)            --                 --
Calcium (mg/dL)                8.55 [ + or -] 0.07     7.6-10.4
Carbon dioxide (mmoUL)                 --
Cholesterol (mg/dL)           316.0 [ + or -] 0           --
Creatine phosphokinase        339.0 [ + or -] 152.7    110-480
 (IU/L)
Creatinine (mg/dL)                     --              0.3-0.4
Gamma glutamyltransferase       5.0 [ + or -] 0          0-2.9
 (mg/dL)
Glucose (mg/dL)               325.0 [ + or -] 32.5     232-269
Lactate dehydrogenase           206 [ + or -] 0         30-205
 (IU/L)
Lipase (IU/L)                  98.0 [ + or -] 0           --
Phosphorus (mg/dL)              3.0 [ + or -] 0        1.8-4.1
Potassium (mmol/L)                     --              3.9-11.7
Sodium (mmol/L)                        --              141-149
Total protein (g/dL)            4.0 [ + or -] 0        2.1-3.3
Triglycerides (mg/dL)         101.0 [ + or -] 0           --
Uric acid (mg/dL)              2.35 [ + or -] 0.35     2.5-12.9

(a) Reference ranges from Carpenter, JW (ed). Exotic Animal
Formulary. 3rd ed. St Louis, MO: Elsevier Saunders; 2005.

(b) No eosinophils or basophils were identified in any of the
blood smears.

Table 2. Results of hematologic and plasma biochemical analysis
(mean [+ or -] SD) of white Carneaux pigeons after oral
administration of aflatoxin B, (3 mg/kg) given once daily for 2,
3, or 4 consecutive days (groups A, D, or B, respectively).

Parameter                          Group A

Hematocrit (%)                51.8 [+ or -] 4.6
Red blood cells (x            3.96 [+ or -] 0.53
 [10.sup.6]/[micro]L)
White blood cells (x         20.74 [+ or -] 8.6
 [10.sup.3]/[micro]L) (c)
  Heterophils (%)             71.8 [+ or -] 17.5
  Lymphocytes (%)             27.6 [+ or -] 17.4
  Monocytes (%)                0.8 [+ or -] 0.8
Alanine aminotransferase     205.0 [+ or -] 169.3
 (IU/L)
Amylase (IU/L)                1571 [+ or -] 1902
Aspartate aminotransferase     930 [+ or -] 12506
 (IU/L)
Bile acids ([micro]mol/L)    46.78 [+ or -] 48.0
Blood urea nitrogen            3.0 [+ or -] 0.7
 (mg/dL)
Calcium (mg/dL)               8.62 [+ or -] 1.51
Carbon dioxide (mmol/L)      28.67 [+ or -] 2.08
Cholesterol (mg/dL)          195.8 [+ or -] 28.0
Creatine phosphokinase       390.4 [+ or -] 131.8
 (IU/L)
Creatinine (mg/dL)            0.36 [+ or -] 0.19
Gamma glutamyltransferase      7.0 [+ or -] 2.3
 (mg/dL)
Glucose (mg/dL)              285.6 [+ or -] 39.3
Lactate dehydrogenase         3174 [+ or -] 5122
 (IU/L)
Lipase (IU/L)                 1224 [+ or -] 1440
Phosphorus (mg/dL)            4.72 [+ or -] 4.64
Potassium (mmol/L)             2.0 [+ or -] 0.6
Sodium (mmol/L)             131.33 [+ or -] 3.05
Total protein (g/dL)          3.28 [+ or -] 0.67
Triglycerides (mg/dL)         68.6 [+ or -] 13.2
Uric acid (mg/dL)              5.2 [+ or -] 3.3

Parameter                            Group D

Hematocrit (%)                 50.0 [+ or -] 4.3
Red blood cells (x             3.86 [+ or -] 0.68
 [10.sup.6]/[micro]L)
White blood cells (x          28.03 [+ or -] 6.02 (d)
 [10.sup.3]/[micro]L) (c)
  Heterophils (%)              86.0 [+ or -] 1.63 (b)
  Lymphocytes (%)             11.75 [+ or -] 1.7 (b)
  Monocytes (%)                2.25 [+ or -] 0.5 (b)
Alanine aminotransferase     226.33 [+ or -] 57.7
 (IU/L)
Amylase (IU/L)                  699 [+ or -] 307
Aspartate aminotransferase      879 [+ or -] 120 9 (d)
 (IU/L)
Bile acids ([micro]mol/L)     89.75 [+ or -] 53.8 (d)
Blood urea nitrogen             6.0 [+ or -] 1
 (mg/dL)
Calcium (mg/dL)                7.68 [+ or -] 0.12
Carbon dioxide (mmol/L)        30.0 [+ or -] 4.3
Cholesterol (mg/dL)          218.25 [+ or -] 27.0 (b)
Creatine phosphokinase        442.2 [+ or -] 142.8
 (IU/L)
Creatinine (mg/dL)             0.46 [+ or -] 0.11
Gamma glutamyltransferase      10.5 [+ or -] 4.7
 (mg/dL)
Glucose (mg/dL)               332.7 [+ or -] 104.0
Lactate dehydrogenase          4358 [+ or -] 2243 (d)
 (IU/L)
Lipase (IU/L)                   427 [+ or -] 254
Phosphorus (mg/dL)             4.48 [+ or -] 1.85 (b)
Potassium (mmol/L)             3.57 [+ or -] 1.02
Sodium (mmol/L)              129.67 [+ or -] 8.73
Total protein (g/dL)           2.65 [+ or -] 0.66
Triglyceride (mg/dL)           85.0 [+ or -] 38.2
Uric acid (mg/dL)              3.98 [+ or -] 1.62 (b)

                                                        Reference
Parameter                            Group B            range (a)

Hematocrit (%)                  51.0 [+ or -] 4.26      39.3-59.4
Red blood cells (x              3.62 [+ or -] 0.09       2.1-4.2
 [10.sup.6]/[micro]L)
White blood cells (x           15.05 [+ or -] 3.88        10-30
 [10.sup.3]/[micro]L) (c)
  Heterophils (%)               86.5 [+ or -] 0.7         15-50
  Lymphocytes (%)               10.5 [+ or -] 0.7         25-70
  Monocytes (%)                  3.0 [+ or -] 1.4 (b)      1-3
Alanine aminotransferase       129.0 [+ or -] 32.5        19-48
 (IU/L)
Amylase (IU/L)                   435 [+ or -] 11            --
Aspartate aminotransferase       671 [+ or -] 140 (d)     45-123
 (IU/L)
Bile acids ([micro]mol/L)       67.7 [+ or -] 37.6        22-60
Blood urea nitrogen              3.0 [+ or -] 0             --
 (mg/dL)
Calcium (mg/dL)                 8.35 [+ or -] 0.63       7.6-10.4
Carbon dioxide (mmol/L)         28.5 [+ or -] 0.7           --
Cholesterol (mg/dL)           230.0  [+ or -] 28.2 (b)       --
Creatine phosphokinase         494.5 [+ or -] 251        110-480
 (IU/L)
Creatinine (mg/dL)              0.35 [+ or -] 0.21       0.3-0.4
Gamma glutamyltransferase       13.0 [+ or -] 4.2          0-2.9
 (mg/dL)
Glucose (mg/dL)                354.0 [+ or -] 118.7      232-269
Lactate dehydrogenase           3713 [+ or -] 752 (b)     30-205
 (IU/L)
Lipase (IU/L)                    135 [+ or -] 38            --
Phosphorus (mg/dL)               5.5 [+ or -] 2.5 (d)    1.8-4.1
Potassium (mmol/L)               3.7 [+ or -] 0.98       3.9-4.7
Sodium (mmol/L)                127.5 [+ or -] 2.1        141-149
Total protein (g/dL)             2.2 [+ or -] 0 (b)      2.1-3.3
Triglycerides (mg/dL)           51.5 [+ or -] 4.9           --
Uric acid (mg/dL)               8.25 [+ or -] 0.77 (b)   2.5-12.9

(a) Reference ranges from Carpenter, JW (ed). Exotic Animal
Formulary. 3rd ed. St Louis, MO: Elsevier Saunders; 2005.

(b) P values <.05 compared with values in Table 1.

(c) No eosinophils or basophils were identified in any of the
blood smears.

(d) P values <.01 compared with values in Table 1.

Table 3. Results of hepatobiliary scintigraphy response
measurements (mean [+ or -] SD) of white Carneaux pigeons before
and after oral administration of aflatoxin B1 as described in
Tables 1 and 2. Scintigraphic images were acquired immediately
after injection of technetium 99m mebrofenin (time = 0) and for a
total acquisition time of 40 minutes.

Parameter   Group A (n = 5)            Group D (n = 4)

AUCh calculated (counts/mL X min)

  Pre      9509 [+ or -] 5616       9505 [+ or -] 5476
  Post   19 883 [+ or -] 22 748   67 562 [+ or -] 43 316 (a)

AUCh trapezoid (counts/mL X min)

  Pre      5951 [+ or -] 3212       5632 [+ or -] 3167
  Post     9841 [+ or -] 7956      20488 [+ or -] 6972 (a)

Fast phase [T.sub.1/2] plasma clearance (min)

  Pre      0.77 [+ or -] 0.09       0.75 [+ or -] 0.04
  Post     1.21 [+ or -] 0.37       2.87 [+ or -] 1.3 (a)

Hepatic excretion [T.sub.1/2] in min)

  Pre      6.65  [+ or -] 2.61     11.95 [+ or -] 9.35
  Post    114.78 [+ or -] 211      184.6 [+ or -] 258.8

Hepatic extraction fraction (%)

  Pre      79.2 [+ or -] 36.4      60.68 [+ or -] 34.15
  Post    82.84 [+ or -] 28.87     99.17 [+ or -] 6.69

  Slow phase [T.sub. 1/2 plasma clearance (min)

  Pre     18.44 [+ or -] 6.28      21.11 [+ or -] 6.75
  Post    25.88 [+ or -] 17.39     68.67 [+ or -] 44.34 (b)

  Time to maximum liver uptake (min)

  Pre      2.75 [+ or -] 0.57       3.19 [+ or -] 1.4
  Post     4.48 [+ or -] 2.44       7.96 [+ or -] 3.18 (a)

Parameter   Group B (n = 2)

AUCh calculated (counts/mL X min)

  Pre      5426 [+ or -] 766
  Post   86 797 [+ or -] 17 868 (a)

AUCh trapezoid (counts/mL X min)

  Pre      3514 [+ or -] 686
  Post   24 351 [+ or -] 1115 (a)

Fast phase [T.sub.1/2] plasma clearance (min)

  Pre       0.8 [+ or -] 0.18
  Post    3.99  [+ or -] 1.6 (a)

Hepatic excretion [T.sub.1/2] in min)

  Pre      6.94 [+ or -] 0.33
  Post   174.49 [+ or -] 65.56

Hepatic extraction fraction (%)

  Pre      55.8 [+ or -] 3.6
  Post     97.3 [+ or -] 2.8

  Slow phase [T.sub. 1/2 plasma clearance (min)

  Pre      14.1 [+ or -] 1.8
  Post     86.2 [+ or -] 18.7 (a)

  Time to maximum liver uptake (min)

  Pre      3.21 [+ or -] 0.65
  Post    10.25 [+ or -] 0.82 (a)

Abbreviation: AUCh indicates area under the heart
time-activity curve.

(a) P<.01.

(b) P < .05.

Table 4. Histopathologic scores (mean [+ or -] SD) of hepatic
biopsy specimens from white Carneaux pigeons before and after
aflatoxin [B.sub.1] administration as described in Tables 1 and
2. Scores were assigned based on the amount of abnormal hepatic
tissue observed: 0 = no abnormal tissue, 1 = 1%-10% of tissue
abnormal, 2 = 11%-25% of tissue abnormal, 3 = 26%-74% of tissue
abnormal, and 4 = 75%-100% of tissue abnormal.

                             Group A (n = 5)

Parameter              Pre                  Post

Degeneration       0 [+ or -] 0       2 [+ or -] 0 (a)
Necrosis           0 [+ or -] 0     1.6 [+ or -] 1.5 (a)
Inflammation     0.6 [+ or -] 0.8     2 [+ or -] 0 (a)
Hemorrhage         0 [+ or -] 0     0.4 [+ or -] 0.8
Overall grade    0.6 [+ or -] 0.8   6.0 [+ or -] 1.8 (a)

                            Group D (n = 4)

Parameter              Pre                 Post

Degeneration      0 [+ or -] 0       2.0 [+ or -] 0 (a)
Necrosis          0 [+ or -] 0      1.25 [+ or -] 0.5 (b)
Inflammation    1.5 [+ or -] 1      2.25 [+ or -] 0.5
Hemorrhage        0 [+ or -] 0         0 [+ or -] 0
Overall grade   1.5 [+ or -] 1       5.5 [+ or -] 0.5 (a)

                            Group B (n = 2)

Parameter              Pre                  Post

Degeneration       0 [+ or -] 0     2.5 [+ or -] 0.7 (a)
Necrosis           0 [+ or -] 0     1.0 [+ or -] 0
Inflammation     1.5 [+ or -] 0.7   2.0 [+ or -]  0
Hemorrhage         0 [+ or -] 0       0 [+ or -] 0
Overall grade    1.5 [+ or -] 0.7   5.5 [+ or -] 0.7 (a)

(a) P < .01.

(b) P < .05.
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Article Details
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Title Annotation:Original Studies
Author:Hadley, Tarah L.; Grizzle, Judith; Rotstein, David S.; Perrin, Shannon; Gerhardt, Lillian E.; Beam,
Publication:Journal of Avian Medicine and Surgery
Article Type:Report
Geographic Code:1USA
Date:Sep 1, 2010
Words:7336
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