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Effect of Dietary Aflatoxin on the Uptake and Elimination of Chlortetracycline in Broiler Chicks1
ENVIRONMENT AND HEALTH Effect of Dietary Aflatoxin on the Uptake and Elimination of Chlortetracycline in Broiler Chicks1 B. L. MILLER and R. D. WYATT2 Department of Poultry Science, University of Georgia, Athens, Georgia 30602 (Received for publication February 6, 1984)
1985 Poultry Science 64:1637-1643 INTRODUCTION
The concentration of many plasma constituents is reduced during aflatoxicosis. Proteins (Tung et al, 1975), lipids (Tung et al, 1972), vitamins (Lynd and Lynd, 1971; Voight et al., 1980), and pigments (Tung and Hamilton, 1973) have all been shown to be significantly reduced in chickens receiving dietary aflatoxin. Voight et al. (1980) suggested that the decrease in free amino acids and B vitamins in the plasma of birds receiving aflatoxin was perhaps partially due to a decrease in their absorption rates. However, Ruff and Wyatt (1976) found that aflatoxicosis did not affect the in vitro intestinal absorption of L-methionine and glucose. A major concern during aflatoxicosis is the occurrence of infections from opportunistic pathogens. For example, Brown and Abrams (1965) were able to isolate Salmonella more frequently from birds with aflatoxicosis.
1
Supported by State and Hatch funds allocated to the Georgia Agricultural Experiment Stations of the University of Georgia. 2 To whom correspondence should be addressed.
Hamilton and Harris (1971) also found a significant interaction on body weights and crop weights of birds that were receiving dietary aflatoxin and infected with Candida albicans. This increased susceptibility to infections could be attributed to the adverse effects of aflatoxin on the immune system. Tung et al. (1975) found aflatoxin to significantly reduce the IgG component of serum, and Giambrone et al. (1978) demonstrated that both the IgG and IgA components were reduced. Giambrone et al. (1978) also demonstrated that chickens fed aflatoxin were deficient in cell-mediated immunity as measured by delayed-type hypersensitivity and graft vs. host reaction. Aflatoxin also suppressed hemagglutinin formation in chickens and thereby depressed the primary immune response (Thaxton et al, 1974). Chlortetracycline is a preventive and therapeutic antibiotic in poultry. It has been shown effective in controlling signs of infectious synovitis (Olson and Sahu, 1976), in reducing numbers of Clostridium perfringens isolated from the ceca of birds infected with Eimeria tenella (Arakawa and Ohe, 1975) and in reducing shed of Salmonella typhimurium in turkey poults (Nivas et al, 1976).
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ABSTRACT Day-old broiler chicks were fed 0, 1.25, 2.5, and 5.0 jug aflatoxin/g of feed for 3 weeks. Chlortetracycline (CTC) was administered in the drinking water at a concentration of 50 mg/liter for 27 hr starting on Day 21. Three hours after initiation of the CTC treatment, birds receiving 2.5 and 5.0 Mg/g dietary aflatoxin had significantly lower CTC in blood compared with the controls. At 12 and 27 hr after the initiation of CTC treatment, birds receiving any level of aflatoxin had significantly decreased blood levels of CTC compared with the controls. Intravenous injection of CTC of control birds and birds receiving 2.5 Mg/g aflatoxin revealed a significant decrease in the elimination half-life of CTC and a significant increase in the total systemic clearance of CTC in the birds receiving dietary aflatoxin. Birds receiving 2.5 Mg/g dietary aflatoxin also had a significant increase in the volume of gall bladder bile; however, this did not result in a greater amount of CTC being eliminated via bile. Studies on protein binding of CTC in the plasma of control birds and the plasma of birds receiving 2.5 Mg/g dietary aflatoxin demonstrated that 60% more CTC is "free", or unbound, in the plasma of birds receiving no aflatoxin. These results suggest that aflatoxicosis lowers the plasma concentrations of CTC as a result of a decreased binding of CTC to plasma protein. This allows more unbound CTC to be available for elimination from the plasma by a means other than the liver, most likely via the kidney. (Key words- chlortetracycline, bile, aflatoxicosis, broilers)
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MILLER AND WYATT
Because aflatoxin does increase the susceptibility of birds to infections, antibiotics would likely be given to birds experiencing an infection along with an aflatoxicosis. However, absorption of the antibiotic may be hampered due to aflatoxicosis. This study was conducted to determine the effect of dietary aflatoxin on blood levels of chlortetracycline (CTC) in poultry and investigate the hypothesized interference of CTC absorption. MATERIALS AND METHODS
The plasma CTC concentration was plotted against time on semilogarithmic graph paper. An average elimination curve was calculated for both groups of birds. The slope and intercept of the elimination curve for each bird were calculated by the method of least squares, and the elimination half-life (t 5 ) was determined from the curve. The total systemic clearance for each bird was calculated using the following equation (Roland and Tozer, 1980): „, .7 X Volume of distribution Clearance = •—• t.s
The remaining 20 birds in each group were killed by cervical dislocation after fasting for 1.5 hr. The cystic bile volume was measured by quantitatively removing the bile from the gall bladder of all birds using a 25-gauge needle and 1-ml syringe. One-tenth milliliter of each bile sample was added to 3 ml of water, and the optical density of the resulting solution was determined at 628 nm. Experiment 3 was conducted to determine the amount of CTC bound to proteins in the plasma of birds experiencing aflatoxicosis compared with the amount of binding oc-
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Male broiler chicks (Hubbard X Hubbard) were used in all experiments and were obtained from a commercial hatchery at day of age. They were housed in electrically heated battery brooders under continuous illumination with feed and water available ad libitum. The aflatoxin used in this study was produced by growing Aspergillus parasiticus NRRL 2999 on sterile polished rice by the method of Shotwell et al. (1966) as modified by West et al. (1973). The moldy rice was dried in a forced air oven and then ground to a fine powder. The rice powder was then analyzed for aflatoxin content by the method of Nabney and Nesbitt (1965) as modified by Wiseman et al. (1967). Chlortetracycline'HC1 was used in this study (Sigma Chemical Company, St. Louis, MO). All plasma from Experiments 1, 2, and 3 was assayed for CTC in triplicate by a microbiological method (Miller and Wyatt, unpublished data). In Experiment 1, weighed amounts of aflatoxin-contaminated rice powder were incorporated into the University of Georgia unmedicated broiler starter diet to achieve dietary levels of 0, 1.25, 2.5, and 5.0 ng aflatoxin/g of feed. Each diet was fed to three replicate groups of 10 chicks from day-old to 3 weeks of age. Body weights were measured weekly. When birds were 3 weeks of age, CTC was added to the drinking water at 50 mg/liter. At 3, 12, and 27 hr after initiation of CTC treatment, a .45-ml sample of blood was taken from the wing vein of each bird into a syringe containing 0.5 ml of .2 M sodium citrate. After 27 hr of CTC administration, CTC was removed, and fresh water was added to the drinkers. Three hours later, another blood sample was taken. Plasma was obtained by centrifugation and frozen at —60 C until analyzed for CTC the following day. At the 3-, 12-, and 27-hr bleedings, water consumption was measured for the previous time interval for
each replicate group. After the final bleeding, the birds were killed by cervical dislocation; and livers were excised, blotted, and weighed. The crude lipid content of each liver was determined gravimetrically by the method of Smith and Hamilton (1970). A second experiment was conducted to determine if the decreased plasma concentrations of CTC were indeed a result of decreased absorption. Experiment 2 consisted of two groups of 40 birds each. One group received the University of Georgia unmedicated broiler starter diet from day-old to 3 weeks of age and served as the controls. The other group received the same basal diet containing 2.5 /Ug aflatoxin/g of feed. At 3 weeks of age, 20 birds from each group were injected intravenously with an aqueous solution of CTC (300 jUg CTC/kg body weight). Blood was taken from each bird at 10, 20, 40, and 80 min after injection as previously described. The plasma was obtained by centrifugation and frozen at —60 C until analyzed for CTC the following day. The plasma was also analyzed for total protein by the method of Wooton (1964) and for albumin by the method of Doumas et al. (1971). The globulin fraction was calculated as the difference between the total protein content and the albumin fraction.
AFLATOXICOSIS AND CHLORTETRACYCLINE
(CTC in plasma) - (CTC in dH 2 Q) (CTC in plasma)
- ._
The concentration of CTC in plasma and the concentration of CTC in d H 2 0 used in the equation are those concentrations that gave the same inhibition zone diameter. Experiment 4 was conducted to determine the effect of dietary aflatoxin on the elimination of CTC via the bile. Two groups of 10 birds each were used in this experiment. One group had received the University of Georgia unmedicated broiler starter diet from day-old to 3 weeks and the other group received the same basal ration containing 2.5 jug aflatoxin/g feed from day-old to 3 weeks. At 3 weeks of age, all birds were injected intravenously with an aqueous solution of CTC at a level of 500 /ig CTC/kg body weight after fasting for 1 hr. Thirty minutes after injection, the birds were killed by cervical dislocation; bile was collected from individual birds as in Experiment 2. The cystic bile volume was measured, and each bile sample was assayed for CTC by the fluorometric method of Kohn (1961). All data were subjected to an analysis of variance in which an F-ratio was calculated. If the F-ratio was significant, the least significant difference among the treatment means was calculated (Bruning and Kintz, 1968). Statements of significance are based on P<.05.
RESULTS
Dietary aflatoxin produced significant decreases in 3-week body weights and significant increases in relative liver weights and liver lipid (Table 1). Relative liver weights and liver lipid were the most sensitive indicators of aflatoxicosis with significant increases at 2.5 and 5.0 /ig/g of dietary aflatoxin. Three-week body weight was significantly decreased only at the highest level of dietary aflatoxin. Three hours after initiation of CTC treatment, birds receiving 2.5 and 5.0£tg/g dietary aflatoxin had significantly lower plasma CTC concentrations compared with the birds receiving no dietary aflatoxin (Fig. 1). Although the birds receiving the lowest dietary aflatoxin showed no significant depression of plasma CTC levels at 3 hr, their plasma concentrations were reduced by 24.4% from the plasma concentrations of the control birds. After 12 and 27 hr of CTC administration, all levels of dietary aflatoxin had significantly lowered plasma CTC concentrations when compared with the controls. When CTC was removed from the drinking water and a blood sample taken 3 hr later, the birds receiving the highest aflatoxin had significantly lower plasma CTC concentrations than the controls and the birds receiving 1.25 Hg/g dietary aflatoxin. The birds receiving 2.5 jug/g dietary aflatoxin had significantly lower CTC concentrations when compared only with the controls. The disappearance rate of chlortetracycline from the plasma of all treatments appeared similar.
TABLE 1. Effect of dietary aflatoxin on 3-vjeek body •weights, relative liver weights, and liver lipid of broiler chicks (Experiment 1) Dietary 3-Week aflatoxin body weight
Liver weight
Liver lipid
(Mg/g) 0 1.25 2.5 5.0
(g/100gBW) 2
(% of DM)
(g) 543 524 498 408
± ± + ±
a
10 >' 32 a 19 a 6b
2.82 2.89 .3.20 4.39
± .04 a ± .04 a ± .08 b ± .14 c
15.5 17.4 24.2 34.9
+ .la ± .3a + .lb ± .4 C
' ' Means within the same column with different superscripts are significantly different (P<.05). 1
Mean ± SEM.
2
BW = Body weight; DM = dry matter.
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curring in the plasma of birds that had received no aflatoxin. In this experiment, .9 ml of blood were obtained from the wing vein of 10 birds that had received the University of Georgia unmedicated broiler starter diet for 3 weeks and from 10 birds that had received the same basal diet containing 2.5 £ig aflatoxin/g of feed for 3 weeks. The .9-ml blood sample was collected into a 1.0 ml syringe containing .1 ml of .2 M sodium citrate. The citrated blood from each bird within a group was pooled to obtain two samples, normal blood and aflatoxin blood. Plasma was obtained by centrifugation of the two pooled samples. Standards containing .05, .10, .25, and .50 jug CTC/ml were prepared using the normal plasma, the aflatoxin plasma, and deionized water ( d H 2 0 ) . These standards were allowed to incubate at room temperature for 30 min for binding and then were assayed for CTC in triplicate. Drug binding percentage was calculated by the following equation (Craig and Suh, 1980):
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MILLER AND WYATT
.120 r
FIG. 1. Effect of dietary aflatoxin on plasma concentrations of chlortetracycline (Experiment 1).
Birds receiving 2.5 Mg/g dietary aflatoxin and administered CTC for 3 hr had a significantly lower CTC intake than the controls and the birds receiving 1.25 /ig/g dietary aflatoxin (Table 2). From 3 to 12 hr of CTC administration, the birds receiving the highest afla-
TABLE 2. Effect of dietary aflatoxin on chlortetracycline (CTC) intake in broiler chicks (Experiment 1) CTC intake
Dietary aflatoxin 0-3 hr (Mg/g) 0 1.25 2.5 5.0
3-12 hr
12-27 hr
- (Mg/kg/hr) 705 717 563 623
a
± 47.7 . 703 ± ±44.1a 707 + ± 18.6 b 643 ± ab + 24.6 577 ±
22.0 3 39.2 a 6.01ab 34.8 b
632 662 585 549
+ ± ± ±
49.1ab 20.9 a 12.6 a b 31.2 b
a ' b Means within the same column with different superscripts are significantly different (P<.05). 1
TABLE 3. Effect of dietary aflatoxin on plasma chlortetracycline (CTC) levels — corrected for decreased intake (Experiment 1)
Mean ± SEM.
Dietary aflatoxin (Mg/g) 1.25 2.5 5.0
CTC in plasma 3hr
12 hr
27 hr
(% of controls)73.9 77.8 70.6
75.6 67.5 73.0
70.8 68.6 67.2
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10 20 TIME (hours)
toxin had a significantly lower CTC intake compared with the controls and birds receiving 1.25 /Ug/g dietary aflatoxin. No aflatoxin treatment produced a significant reduction in CTC intake compared to the controls from 12 to 27 hr of CTC administrations; however, within this time interval the birds receiving 5.0 jUg/g dietary aflatoxin did have a significantly reduced CTC intake when compared with the birds receiving the lowest level of aflatoxin. The reductions in CTC intake are due to an aflatoxin-induced decrease in water consumption. When the percent decrease in CTC intake was subtracted from the percent decrease in plasma CTC concentration, the birds receiving dietary aflatoxin still had substantially decreased plasma CTC concentrations compared to the controls (Table 3). In Experiment 2, the birds receiving aflatoxin in their diet exhibited typical responses with regard to the protein components of plasma. Total plasma protein, plasma albumin and plasma globulin were all significantly reduced in these birds compared to the birds receiving no aflatoxin in their diet (Table 4). The slope of the curve for the birds receiving aflatoxin is increased by 87% over the slope of the curve for the control birds (Fig. 2). Calculation of t. 5 and total systemic clearance rate from these data clearly indicates increased clearance of CTC from the plasma of birds with aflatoxicosis in relation o control birds (Table 5). The birds receiving dietary aflatoxin in Experiment 2 had an average volume of bile in the gall bladder of 1.26 m£/kg following a 1.5-hr fast (Table 6). This amount was a significant increase in gall bladder bile volume over the volume of .66 ml/kg for the control birds. Biliary pigmentation was also significantly reduced in the birds receiving dietary
AFLATOXICOSIS AND CHLORTETRACYCLINE TABLE 4. Effect of dietary aflatoxin on proteinaceous components of blood plasma in broiler chicks (Experiment 2) Dietary Total aflatoxin protein
,
Albumin
,>
(Mg/g) 0 2.5
2.55 ± .05a>' 1.37 ± . l i b
Dietary aflatoxin
Elimination half-life
Total systemic clearance
(Mg/g) 0 2.5
(min)
(m£/min)
1.58 ± .05 a .78 + .08 b
34.8 ± 1.5a>' 22.8 ± 1.2b
21.4 ± 1.0a 39.6 + 3.0 b
a ' b Means within the same column with different superscripts are significantly different (P=S.05). 1
TABLE 5. Effect of dietary aflatoxin on the elimination half-life and total systemic clearance of chlortetracycline in broiler chicks (Experiment 2)
Globulin
(g/100 g) .97 + .02 a .59 ± .04 b
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a ' Means within the same column with different superscripts are significantly different (P<.05). 1
Mean ± SEM.
DISCUSSION
Decreased absorption from the intestinal tract has been suggested as the cause of lowered concentrations of plasma carotenoids (Tung and Hamilton, 1973) and free amino acids (Voight et al, 1980) during aflatoxicosis. Hamilton et al. (1974) showed a significant interaction between riboflavin and vitamin D 3 deficiencies and aflatoxicosis in broiler chicks and suggested that aflatoxin impairs riboflavin absorption. In the present investigation, the data from Experiment 1 demonstrated that dietary aflatoxin significantly lowers the plasma concentration of CTC. The disappearance rate of CTC from the plasma appeared to be similar for all treatments, therefore suggesting that the decrease in plasma CTC could be due to a decrease in the absorption of the drug from the gastro-intestinal tract. However, the decreased t. 5 and increased total systemic clearance rate clearly demonstrate that the decreased plasma
TABLE 6. Effect of dietary aflatoxin on bile volume and concentration (Experiment 2)
25
50
75
Dietary aflatoxin
Bile volume
Bile concentration
(Mg/g) 0 2.5
(mK/kg)
(o.d. @628nm)
.66 + .06a>' 1.26 ± .15 b
.336 ± .022 a .149 ± .018 b
100
TIME (minutes) FIG. 2. Average elimination curve of chlortetracycline from plasma of broiler chicks (Experiment 2).
ab ' Means within the same column with different superscripts are significantly different (P<.05). 1
Mean ± SEM.
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aflatoxin suggesting a dilution effect due to increased bile volume (Table 6). In Experiment 4, aflatoxicosis again resulted in a significant increase in bile volume (Table 7); however, there was no significant difference in the total amount of CTC found in the cystic bile of the birds receiving aflatoxin and the control birds. From the data presented in Figure 3, it can be determined that in normal plasma, which had a total protein content of 2.55 g/100 ml, 50% of the CTC present is bound to plasma proteins. Binding of CTC to proteins in plasma from birds with aflatoxicosis having a total protein content of only 1.00 g/100 ml appeared to be only 20%.
Mean + SEM.
MILLER AND WYATT
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TABLE 7. Effect of dietary aflatoxin on bile volume and total chlortetracycline (CTC) content of the bile (Experiment 4) Dietary aflatoxin
Bile volume
Total cystic CTC
(Mg/g) 0 2.5
(mB/kg)
(
.38+ .06 a ." .85 + . 2 1 b
a ' b Means within the same column with different superscripts are significantly different (P<.05). 1
Mean ± SEM.
o
6
10
14
18
INHIBITION ZONE DIAMETER(mm)
FIG. 3. Effect of dietary aflatoxin on the binding of chlortetracycline in the plasma of broiler chicks (Experiment 3).
' ' Points with different superscripts along the same concentration of chlortetracycline are significantly different (P<.05).
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CTC concentrations are a result of an increased elimination of the drug from the blood rather than due to poor intestinal uptake. Aflatoxin produced a significant decrease in the t. 5 of 34% and a significant increase in the total systemic clearance of 85%. Witlock et al. (1977) demonstrated that aflatoxin did not alter the gross morphology of any region of the avian intestinal tract. Ruff and Wyatt (1976) suggested that aflatoxin might bring about decreases in blood components through physiological mechanisms other than malabsorption. All of the tetracyclines are eliminated from the body via the liver and kidney with the majority of the drug being eliminated in the urine. Chlortetracycline is more dependent on biliary excretion for its elimination than are the other tetracyclines (Sande and Mandell, 1980). Kunin et al. (1959) found in humans that CTC was removed from the blood by nonrenal mechanisms at a rate of 9.5%/hr compared to a rate of 3 to 4%/hr for the other tetracyclines. Because aflatoxin was found to increase biliary output, it was of interest in the present study to determine if this might increase the biliary excretion of CTC and thereby account for lowered plasma concentrations of CTC. The results of Experiment 4 show that the increased biliary output produced by the aflatoxicosis did not alter biliary elimination of CTC. The concentration of CTC in the bile was reduced in the birds receiving dietary aflatoxin due to the increased volume of bile in these birds diluting the total amount of CTC present in the gall bladder lumen. However, the quantity of CTC found in the gall bladder of controls and birds with aflatoxicosis was not different. This result is in agreement with an earlier report by Zaslow et al. (1955) in which dehydrocholic acid, a
powerful choleretic, decreased the concentration of tetracycline and oxytetracycline in the bile of humans. Chlortetracycline is filtered at the glomerulus and is neither reabsorbed nor secreted by the kidney tubules (Sirota and Saltzman, 1950). Kunin et al. (1959) found that CTC was bound to approximately twice the extent as tetracycline and oxytetracycline in human plasma and also that the renal clearance of CTC was only half the renal clearance of tetracycline and oxytetracycline. Therefore, these workers demonstrated that the rate of renal excretion of the tetracyclines is inversely related to the amount of protein binding that occurs within the plasma. This same phenomenon has also been observed with the anticoagulant, warfarin (Levy and Yocobi, 1974). Therefore, only the fraction of drug that is "free", or unbound, in the plasma is available to be eliminated via the urine. The quantitative binding of a drug to plasma proteins can be affected by many factors. One such factor is the concentration of proteins in the plasma. It is a well-established fact that aflatoxin produces a significant hypoproteinemia in broiler chicks. In the present investigation, plasma from birds receiving aflatoxin demonstrated only 40% of the binding that occurred with plasma from birds not receiving aflatoxin. With this reduced binding of CTC present in the plasma of birds experiencing
AFLATOXICOSIS AND CHLORTETRACYCLINE
aflatoxicosis, the renal clearance of the drug is apparently increased. On the basis of the present investigation, aflatoxin reduces the plasma concentration of CTC due to an increased elimination of the drug from the plasma. This increased elimination appears to be the result of a decreased binding of CTC to plasma proteins, thus allowing more unbound CTC to be available for elimination from the plasma by a means other than the liver, most likely via the kidney. REFERENCES
the diet of turkey poults artifically-infected with Salmonella typhimurium. Poultry Sci. 55: 2176-2189. Olson, N. O., and S. P. Sahu, 1976. Efficacy of chlortetracycline against Mycoplasma synoviae isolated in two periods. Avian Dis. 20:221—229. Rowland, M., and T. N. Tozer, 1980. Clinical Pharmacokinetics: Concepts and Applications. Lea and Febiger, Philadelphia, PA. Ruff, M. D., and R. D. Wyatt, 1976. Intestinal absorption of L-methionine and glucose in chickens with aflatoxicosis. Toxicol. Appl. Pharmacol. 37:257-262. Sande, M. A., and G. L. Mandell, 1980. Antimicrobial agents: Tetracyclines and chloramphenicol. Pages 1181-1199 in The Pharmacological Basis of Therapeutics. 6th ed. A. G. Gilman, L. S. Goodman, and A. Gilman, ed. MacMillian, New York, NY. Shotwell, O. L., C. W. Hesseltine, R. D. Stubblefield, and W. G. Sorenson, 1966. Production of anatoxin on rice. Appl. Microbiol. 14:425—428. Sirota, J. H., and A. Saltzman, 1950. The renal clearance and protein binding of aureomycin in man. J. Pharmacol. Exp. Ther. 100:210-218. Smith, J. W., and P. B. Hamilton, 1970. Aflatoxicosis in the broiler chicken. Poultry Sci. 49:207—215. Thaxton, J. P., H.-T. Tung, and P. B. Hamilton, 1974. Immunosuppression in chickens by aflatoxin. Poultry Sci. 53:721-725. Tung, H.-T., W. E. Donaldson, and P. B. Hamilton, 1972. Altered lipid transport during aflatoxicosis. Toxicol. Appl. Pharmacol. 22:97-104. Tung, H.-T., and P. B. Hamilton, 1973. Decreased plasma carotenoids during aflatoxicosis. Poultry Sci. 5 2 : 8 0 - 8 3 . Tung, H.-T., R. D. Wyatt, P. Thaxton, and P. B. Hamilton, 1975. Concentrations of serum proteins during aflatoxicosis. Toxicol. Appl. Pharmocol. 34:320-326. Voight, M. N., R. D. Wyatt, J. C. Ayers, and P. E. Koehler, 1980. Abnormal concentrations of B vitamins and amino acids in plasma, bile and liver of chicks with aflatoxicosis. Appl. Environ. Microbiol. 40:870-875. West, S., R. D. Wyatt, and P. B. Hamilton, 1973. Improved yield of aflatoxin by incremental increases of temperature. Appl. Microbiol. 25:1018-1019. Witlock, D. R., R. D. Wyatt, and M. D. Ruff, 1977. Morphological changes in the avian intestine induced by citruiin and lack of effect of aflatoxin and T-2 toxin as seen with scanning electron microscopy. Toxicon. 15:41—44. Wiseman, H. G., W. C. Jacobson, andW. C. Harmeyer, 1967. Note on removal of pigments from chloroform extracts of aflatoxin cultures with coppercarbonate. J. Assoc. Offic. Agric. Chem. 50: 982-983. Wooton, I.D.P., 1964. Micro-analysis in Medical Biochemistry. Grune and Stratton, Inc., New York, NY. Zaslow, J., E. M. Cohn, and W. Ball, 1955. The effect of dehydrocholic acid on the excretion and concentration of oxytetracycline and tetracycline in bile. Antibiot. Med. 1:151-152.
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Arakawa, A., and O. Ohe, 1975. Reduction of Clostriddium perfringes by feed additive antibiotics in the ceca of chickens infected with Eimeria tenella. Poultry Sci. 54:1000-1007. Brown, J.M.M., and L. Abrams, 1965. Biochemical studies on aflatoxicosis. Onderstepoort J. Vet. Res. 32:119-146. Bruning, J. L., and B. L. Kintz, 1968. Computational Handbook of Statistics. Scott Foresman Co., Glenview, IL. Craig, W. A., and B. Suh, 1980. Protein binding and the antibacterial effects. Methods for the determination of protein binding, Pages 265—297 in Antibiotics in Laboratory Medicine. Victor Lorian, ed. Williams and Wilkins Co., Baltimore, MD. Doumas, B. T., W. A. Watson, and H. G. Briggs, 1971. Albumin standards and the measurement of serum albumin with bromcresol green. Clin. Chim. Acta 31:87-96. Giambrone, J. J., D. L. Ewert, R. D. Wyatt, and C. S. Eidson, 1978. Effect of aflatoxin on the humoral and cell-mediated immune systems of the chicken. Am. J. Vet. Res. 39:305-308. Hamilton, P. B., and J. R. Harris, 1971. Interaction of aflatoxicosis with Candida albicans infections and other stresses in chickens. Poultry Sci. 50: 906-912. Hamilton, P. B., H.-T. Tung, R. D. Wyatt, and W. E. Donaldson, 1974. Interaction of dietary aflatoxin with some vitamin deficiencies. Poultry Sci. 53:871-877. Kohn, K. W., 1961. Determination of tetracyclines by extraction of fluorescent complexes. Anal. Chem. 33:862-866. Kunin, C. M., A. C. Dornbush, and M. Finland, 1959. Distribution and excretion of four tetracycline analogues in normal young men. J. Clin. Invest. 38:1950-1963. Levy, G., and A. Yacobi, 1974. Effect of plasma protein binding on elimination of warfarin. J. Pharm. Sci. 63:805-806. Lynd, J. Q., and F. T. Lynd, 1971. Mucor divergence in aflatoxin effects with duckling bile and plasma. Environ. Res. 4 : 3 1 6 - 3 2 4 . Nabney, J., and B. F. Nesbitt, 1965. A spectrophotometric method of determining the aflatoxins. Analyst 90:155-160. Nivas, S. C , M. D. York, and B. S. Pomeroy, 1976. Effects of different levels of chlortetracycline in
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1985 Poultry Science 64:1637-1643 INTRODUCTION
The concentration of many plasma constituents is reduced during aflatoxicosis. Proteins (Tung et al, 1975), lipids (Tung et al, 1972), vitamins (Lynd and Lynd, 1971; Voight et al., 1980), and pigments (Tung and Hamilton, 1973) have all been shown to be significantly reduced in chickens receiving dietary aflatoxin. Voight et al. (1980) suggested that the decrease in free amino acids and B vitamins in the plasma of birds receiving aflatoxin was perhaps partially due to a decrease in their absorption rates. However, Ruff and Wyatt (1976) found that aflatoxicosis did not affect the in vitro intestinal absorption of L-methionine and glucose. A major concern during aflatoxicosis is the occurrence of infections from opportunistic pathogens. For example, Brown and Abrams (1965) were able to isolate Salmonella more frequently from birds with aflatoxicosis.
1
Supported by State and Hatch funds allocated to the Georgia Agricultural Experiment Stations of the University of Georgia. 2 To whom correspondence should be addressed.
Hamilton and Harris (1971) also found a significant interaction on body weights and crop weights of birds that were receiving dietary aflatoxin and infected with Candida albicans. This increased susceptibility to infections could be attributed to the adverse effects of aflatoxin on the immune system. Tung et al. (1975) found aflatoxin to significantly reduce the IgG component of serum, and Giambrone et al. (1978) demonstrated that both the IgG and IgA components were reduced. Giambrone et al. (1978) also demonstrated that chickens fed aflatoxin were deficient in cell-mediated immunity as measured by delayed-type hypersensitivity and graft vs. host reaction. Aflatoxin also suppressed hemagglutinin formation in chickens and thereby depressed the primary immune response (Thaxton et al, 1974). Chlortetracycline is a preventive and therapeutic antibiotic in poultry. It has been shown effective in controlling signs of infectious synovitis (Olson and Sahu, 1976), in reducing numbers of Clostridium perfringens isolated from the ceca of birds infected with Eimeria tenella (Arakawa and Ohe, 1975) and in reducing shed of Salmonella typhimurium in turkey poults (Nivas et al, 1976).
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ABSTRACT Day-old broiler chicks were fed 0, 1.25, 2.5, and 5.0 jug aflatoxin/g of feed for 3 weeks. Chlortetracycline (CTC) was administered in the drinking water at a concentration of 50 mg/liter for 27 hr starting on Day 21. Three hours after initiation of the CTC treatment, birds receiving 2.5 and 5.0 Mg/g dietary aflatoxin had significantly lower CTC in blood compared with the controls. At 12 and 27 hr after the initiation of CTC treatment, birds receiving any level of aflatoxin had significantly decreased blood levels of CTC compared with the controls. Intravenous injection of CTC of control birds and birds receiving 2.5 Mg/g aflatoxin revealed a significant decrease in the elimination half-life of CTC and a significant increase in the total systemic clearance of CTC in the birds receiving dietary aflatoxin. Birds receiving 2.5 Mg/g dietary aflatoxin also had a significant increase in the volume of gall bladder bile; however, this did not result in a greater amount of CTC being eliminated via bile. Studies on protein binding of CTC in the plasma of control birds and the plasma of birds receiving 2.5 Mg/g dietary aflatoxin demonstrated that 60% more CTC is "free", or unbound, in the plasma of birds receiving no aflatoxin. These results suggest that aflatoxicosis lowers the plasma concentrations of CTC as a result of a decreased binding of CTC to plasma protein. This allows more unbound CTC to be available for elimination from the plasma by a means other than the liver, most likely via the kidney. (Key words- chlortetracycline, bile, aflatoxicosis, broilers)
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MILLER AND WYATT
Because aflatoxin does increase the susceptibility of birds to infections, antibiotics would likely be given to birds experiencing an infection along with an aflatoxicosis. However, absorption of the antibiotic may be hampered due to aflatoxicosis. This study was conducted to determine the effect of dietary aflatoxin on blood levels of chlortetracycline (CTC) in poultry and investigate the hypothesized interference of CTC absorption. MATERIALS AND METHODS
The plasma CTC concentration was plotted against time on semilogarithmic graph paper. An average elimination curve was calculated for both groups of birds. The slope and intercept of the elimination curve for each bird were calculated by the method of least squares, and the elimination half-life (t 5 ) was determined from the curve. The total systemic clearance for each bird was calculated using the following equation (Roland and Tozer, 1980): „, .7 X Volume of distribution Clearance = •—• t.s
The remaining 20 birds in each group were killed by cervical dislocation after fasting for 1.5 hr. The cystic bile volume was measured by quantitatively removing the bile from the gall bladder of all birds using a 25-gauge needle and 1-ml syringe. One-tenth milliliter of each bile sample was added to 3 ml of water, and the optical density of the resulting solution was determined at 628 nm. Experiment 3 was conducted to determine the amount of CTC bound to proteins in the plasma of birds experiencing aflatoxicosis compared with the amount of binding oc-
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Male broiler chicks (Hubbard X Hubbard) were used in all experiments and were obtained from a commercial hatchery at day of age. They were housed in electrically heated battery brooders under continuous illumination with feed and water available ad libitum. The aflatoxin used in this study was produced by growing Aspergillus parasiticus NRRL 2999 on sterile polished rice by the method of Shotwell et al. (1966) as modified by West et al. (1973). The moldy rice was dried in a forced air oven and then ground to a fine powder. The rice powder was then analyzed for aflatoxin content by the method of Nabney and Nesbitt (1965) as modified by Wiseman et al. (1967). Chlortetracycline'HC1 was used in this study (Sigma Chemical Company, St. Louis, MO). All plasma from Experiments 1, 2, and 3 was assayed for CTC in triplicate by a microbiological method (Miller and Wyatt, unpublished data). In Experiment 1, weighed amounts of aflatoxin-contaminated rice powder were incorporated into the University of Georgia unmedicated broiler starter diet to achieve dietary levels of 0, 1.25, 2.5, and 5.0 ng aflatoxin/g of feed. Each diet was fed to three replicate groups of 10 chicks from day-old to 3 weeks of age. Body weights were measured weekly. When birds were 3 weeks of age, CTC was added to the drinking water at 50 mg/liter. At 3, 12, and 27 hr after initiation of CTC treatment, a .45-ml sample of blood was taken from the wing vein of each bird into a syringe containing 0.5 ml of .2 M sodium citrate. After 27 hr of CTC administration, CTC was removed, and fresh water was added to the drinkers. Three hours later, another blood sample was taken. Plasma was obtained by centrifugation and frozen at —60 C until analyzed for CTC the following day. At the 3-, 12-, and 27-hr bleedings, water consumption was measured for the previous time interval for
each replicate group. After the final bleeding, the birds were killed by cervical dislocation; and livers were excised, blotted, and weighed. The crude lipid content of each liver was determined gravimetrically by the method of Smith and Hamilton (1970). A second experiment was conducted to determine if the decreased plasma concentrations of CTC were indeed a result of decreased absorption. Experiment 2 consisted of two groups of 40 birds each. One group received the University of Georgia unmedicated broiler starter diet from day-old to 3 weeks of age and served as the controls. The other group received the same basal diet containing 2.5 /Ug aflatoxin/g of feed. At 3 weeks of age, 20 birds from each group were injected intravenously with an aqueous solution of CTC (300 jUg CTC/kg body weight). Blood was taken from each bird at 10, 20, 40, and 80 min after injection as previously described. The plasma was obtained by centrifugation and frozen at —60 C until analyzed for CTC the following day. The plasma was also analyzed for total protein by the method of Wooton (1964) and for albumin by the method of Doumas et al. (1971). The globulin fraction was calculated as the difference between the total protein content and the albumin fraction.
AFLATOXICOSIS AND CHLORTETRACYCLINE
(CTC in plasma) - (CTC in dH 2 Q) (CTC in plasma)
- ._
The concentration of CTC in plasma and the concentration of CTC in d H 2 0 used in the equation are those concentrations that gave the same inhibition zone diameter. Experiment 4 was conducted to determine the effect of dietary aflatoxin on the elimination of CTC via the bile. Two groups of 10 birds each were used in this experiment. One group had received the University of Georgia unmedicated broiler starter diet from day-old to 3 weeks and the other group received the same basal ration containing 2.5 jug aflatoxin/g feed from day-old to 3 weeks. At 3 weeks of age, all birds were injected intravenously with an aqueous solution of CTC at a level of 500 /ig CTC/kg body weight after fasting for 1 hr. Thirty minutes after injection, the birds were killed by cervical dislocation; bile was collected from individual birds as in Experiment 2. The cystic bile volume was measured, and each bile sample was assayed for CTC by the fluorometric method of Kohn (1961). All data were subjected to an analysis of variance in which an F-ratio was calculated. If the F-ratio was significant, the least significant difference among the treatment means was calculated (Bruning and Kintz, 1968). Statements of significance are based on P<.05.
RESULTS
Dietary aflatoxin produced significant decreases in 3-week body weights and significant increases in relative liver weights and liver lipid (Table 1). Relative liver weights and liver lipid were the most sensitive indicators of aflatoxicosis with significant increases at 2.5 and 5.0 /ig/g of dietary aflatoxin. Three-week body weight was significantly decreased only at the highest level of dietary aflatoxin. Three hours after initiation of CTC treatment, birds receiving 2.5 and 5.0£tg/g dietary aflatoxin had significantly lower plasma CTC concentrations compared with the birds receiving no dietary aflatoxin (Fig. 1). Although the birds receiving the lowest dietary aflatoxin showed no significant depression of plasma CTC levels at 3 hr, their plasma concentrations were reduced by 24.4% from the plasma concentrations of the control birds. After 12 and 27 hr of CTC administration, all levels of dietary aflatoxin had significantly lowered plasma CTC concentrations when compared with the controls. When CTC was removed from the drinking water and a blood sample taken 3 hr later, the birds receiving the highest aflatoxin had significantly lower plasma CTC concentrations than the controls and the birds receiving 1.25 Hg/g dietary aflatoxin. The birds receiving 2.5 jug/g dietary aflatoxin had significantly lower CTC concentrations when compared only with the controls. The disappearance rate of chlortetracycline from the plasma of all treatments appeared similar.
TABLE 1. Effect of dietary aflatoxin on 3-vjeek body •weights, relative liver weights, and liver lipid of broiler chicks (Experiment 1) Dietary 3-Week aflatoxin body weight
Liver weight
Liver lipid
(Mg/g) 0 1.25 2.5 5.0
(g/100gBW) 2
(% of DM)
(g) 543 524 498 408
± ± + ±
a
10 >' 32 a 19 a 6b
2.82 2.89 .3.20 4.39
± .04 a ± .04 a ± .08 b ± .14 c
15.5 17.4 24.2 34.9
+ .la ± .3a + .lb ± .4 C
' ' Means within the same column with different superscripts are significantly different (P<.05). 1
Mean ± SEM.
2
BW = Body weight; DM = dry matter.
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curring in the plasma of birds that had received no aflatoxin. In this experiment, .9 ml of blood were obtained from the wing vein of 10 birds that had received the University of Georgia unmedicated broiler starter diet for 3 weeks and from 10 birds that had received the same basal diet containing 2.5 £ig aflatoxin/g of feed for 3 weeks. The .9-ml blood sample was collected into a 1.0 ml syringe containing .1 ml of .2 M sodium citrate. The citrated blood from each bird within a group was pooled to obtain two samples, normal blood and aflatoxin blood. Plasma was obtained by centrifugation of the two pooled samples. Standards containing .05, .10, .25, and .50 jug CTC/ml were prepared using the normal plasma, the aflatoxin plasma, and deionized water ( d H 2 0 ) . These standards were allowed to incubate at room temperature for 30 min for binding and then were assayed for CTC in triplicate. Drug binding percentage was calculated by the following equation (Craig and Suh, 1980):
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MILLER AND WYATT
.120 r
FIG. 1. Effect of dietary aflatoxin on plasma concentrations of chlortetracycline (Experiment 1).
Birds receiving 2.5 Mg/g dietary aflatoxin and administered CTC for 3 hr had a significantly lower CTC intake than the controls and the birds receiving 1.25 /ig/g dietary aflatoxin (Table 2). From 3 to 12 hr of CTC administration, the birds receiving the highest afla-
TABLE 2. Effect of dietary aflatoxin on chlortetracycline (CTC) intake in broiler chicks (Experiment 1) CTC intake
Dietary aflatoxin 0-3 hr (Mg/g) 0 1.25 2.5 5.0
3-12 hr
12-27 hr
- (Mg/kg/hr) 705 717 563 623
a
± 47.7 . 703 ± ±44.1a 707 + ± 18.6 b 643 ± ab + 24.6 577 ±
22.0 3 39.2 a 6.01ab 34.8 b
632 662 585 549
+ ± ± ±
49.1ab 20.9 a 12.6 a b 31.2 b
a ' b Means within the same column with different superscripts are significantly different (P<.05). 1
TABLE 3. Effect of dietary aflatoxin on plasma chlortetracycline (CTC) levels — corrected for decreased intake (Experiment 1)
Mean ± SEM.
Dietary aflatoxin (Mg/g) 1.25 2.5 5.0
CTC in plasma 3hr
12 hr
27 hr
(% of controls)73.9 77.8 70.6
75.6 67.5 73.0
70.8 68.6 67.2
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10 20 TIME (hours)
toxin had a significantly lower CTC intake compared with the controls and birds receiving 1.25 /Ug/g dietary aflatoxin. No aflatoxin treatment produced a significant reduction in CTC intake compared to the controls from 12 to 27 hr of CTC administrations; however, within this time interval the birds receiving 5.0 jUg/g dietary aflatoxin did have a significantly reduced CTC intake when compared with the birds receiving the lowest level of aflatoxin. The reductions in CTC intake are due to an aflatoxin-induced decrease in water consumption. When the percent decrease in CTC intake was subtracted from the percent decrease in plasma CTC concentration, the birds receiving dietary aflatoxin still had substantially decreased plasma CTC concentrations compared to the controls (Table 3). In Experiment 2, the birds receiving aflatoxin in their diet exhibited typical responses with regard to the protein components of plasma. Total plasma protein, plasma albumin and plasma globulin were all significantly reduced in these birds compared to the birds receiving no aflatoxin in their diet (Table 4). The slope of the curve for the birds receiving aflatoxin is increased by 87% over the slope of the curve for the control birds (Fig. 2). Calculation of t. 5 and total systemic clearance rate from these data clearly indicates increased clearance of CTC from the plasma of birds with aflatoxicosis in relation o control birds (Table 5). The birds receiving dietary aflatoxin in Experiment 2 had an average volume of bile in the gall bladder of 1.26 m£/kg following a 1.5-hr fast (Table 6). This amount was a significant increase in gall bladder bile volume over the volume of .66 ml/kg for the control birds. Biliary pigmentation was also significantly reduced in the birds receiving dietary
AFLATOXICOSIS AND CHLORTETRACYCLINE TABLE 4. Effect of dietary aflatoxin on proteinaceous components of blood plasma in broiler chicks (Experiment 2) Dietary Total aflatoxin protein
,
Albumin
,>
(Mg/g) 0 2.5
2.55 ± .05a>' 1.37 ± . l i b
Dietary aflatoxin
Elimination half-life
Total systemic clearance
(Mg/g) 0 2.5
(min)
(m£/min)
1.58 ± .05 a .78 + .08 b
34.8 ± 1.5a>' 22.8 ± 1.2b
21.4 ± 1.0a 39.6 + 3.0 b
a ' b Means within the same column with different superscripts are significantly different (P=S.05). 1
TABLE 5. Effect of dietary aflatoxin on the elimination half-life and total systemic clearance of chlortetracycline in broiler chicks (Experiment 2)
Globulin
(g/100 g) .97 + .02 a .59 ± .04 b
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a ' Means within the same column with different superscripts are significantly different (P<.05). 1
Mean ± SEM.
DISCUSSION
Decreased absorption from the intestinal tract has been suggested as the cause of lowered concentrations of plasma carotenoids (Tung and Hamilton, 1973) and free amino acids (Voight et al, 1980) during aflatoxicosis. Hamilton et al. (1974) showed a significant interaction between riboflavin and vitamin D 3 deficiencies and aflatoxicosis in broiler chicks and suggested that aflatoxin impairs riboflavin absorption. In the present investigation, the data from Experiment 1 demonstrated that dietary aflatoxin significantly lowers the plasma concentration of CTC. The disappearance rate of CTC from the plasma appeared to be similar for all treatments, therefore suggesting that the decrease in plasma CTC could be due to a decrease in the absorption of the drug from the gastro-intestinal tract. However, the decreased t. 5 and increased total systemic clearance rate clearly demonstrate that the decreased plasma
TABLE 6. Effect of dietary aflatoxin on bile volume and concentration (Experiment 2)
25
50
75
Dietary aflatoxin
Bile volume
Bile concentration
(Mg/g) 0 2.5
(mK/kg)
(o.d. @628nm)
.66 + .06a>' 1.26 ± .15 b
.336 ± .022 a .149 ± .018 b
100
TIME (minutes) FIG. 2. Average elimination curve of chlortetracycline from plasma of broiler chicks (Experiment 2).
ab ' Means within the same column with different superscripts are significantly different (P<.05). 1
Mean ± SEM.
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aflatoxin suggesting a dilution effect due to increased bile volume (Table 6). In Experiment 4, aflatoxicosis again resulted in a significant increase in bile volume (Table 7); however, there was no significant difference in the total amount of CTC found in the cystic bile of the birds receiving aflatoxin and the control birds. From the data presented in Figure 3, it can be determined that in normal plasma, which had a total protein content of 2.55 g/100 ml, 50% of the CTC present is bound to plasma proteins. Binding of CTC to proteins in plasma from birds with aflatoxicosis having a total protein content of only 1.00 g/100 ml appeared to be only 20%.
Mean + SEM.
MILLER AND WYATT
1642
TABLE 7. Effect of dietary aflatoxin on bile volume and total chlortetracycline (CTC) content of the bile (Experiment 4) Dietary aflatoxin
Bile volume
Total cystic CTC
(Mg/g) 0 2.5
(mB/kg)
(
.38+ .06 a ." .85 + . 2 1 b
a ' b Means within the same column with different superscripts are significantly different (P<.05). 1
Mean ± SEM.
o
6
10
14
18
INHIBITION ZONE DIAMETER(mm)
FIG. 3. Effect of dietary aflatoxin on the binding of chlortetracycline in the plasma of broiler chicks (Experiment 3).
' ' Points with different superscripts along the same concentration of chlortetracycline are significantly different (P<.05).
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CTC concentrations are a result of an increased elimination of the drug from the blood rather than due to poor intestinal uptake. Aflatoxin produced a significant decrease in the t. 5 of 34% and a significant increase in the total systemic clearance of 85%. Witlock et al. (1977) demonstrated that aflatoxin did not alter the gross morphology of any region of the avian intestinal tract. Ruff and Wyatt (1976) suggested that aflatoxin might bring about decreases in blood components through physiological mechanisms other than malabsorption. All of the tetracyclines are eliminated from the body via the liver and kidney with the majority of the drug being eliminated in the urine. Chlortetracycline is more dependent on biliary excretion for its elimination than are the other tetracyclines (Sande and Mandell, 1980). Kunin et al. (1959) found in humans that CTC was removed from the blood by nonrenal mechanisms at a rate of 9.5%/hr compared to a rate of 3 to 4%/hr for the other tetracyclines. Because aflatoxin was found to increase biliary output, it was of interest in the present study to determine if this might increase the biliary excretion of CTC and thereby account for lowered plasma concentrations of CTC. The results of Experiment 4 show that the increased biliary output produced by the aflatoxicosis did not alter biliary elimination of CTC. The concentration of CTC in the bile was reduced in the birds receiving dietary aflatoxin due to the increased volume of bile in these birds diluting the total amount of CTC present in the gall bladder lumen. However, the quantity of CTC found in the gall bladder of controls and birds with aflatoxicosis was not different. This result is in agreement with an earlier report by Zaslow et al. (1955) in which dehydrocholic acid, a
powerful choleretic, decreased the concentration of tetracycline and oxytetracycline in the bile of humans. Chlortetracycline is filtered at the glomerulus and is neither reabsorbed nor secreted by the kidney tubules (Sirota and Saltzman, 1950). Kunin et al. (1959) found that CTC was bound to approximately twice the extent as tetracycline and oxytetracycline in human plasma and also that the renal clearance of CTC was only half the renal clearance of tetracycline and oxytetracycline. Therefore, these workers demonstrated that the rate of renal excretion of the tetracyclines is inversely related to the amount of protein binding that occurs within the plasma. This same phenomenon has also been observed with the anticoagulant, warfarin (Levy and Yocobi, 1974). Therefore, only the fraction of drug that is "free", or unbound, in the plasma is available to be eliminated via the urine. The quantitative binding of a drug to plasma proteins can be affected by many factors. One such factor is the concentration of proteins in the plasma. It is a well-established fact that aflatoxin produces a significant hypoproteinemia in broiler chicks. In the present investigation, plasma from birds receiving aflatoxin demonstrated only 40% of the binding that occurred with plasma from birds not receiving aflatoxin. With this reduced binding of CTC present in the plasma of birds experiencing
AFLATOXICOSIS AND CHLORTETRACYCLINE
aflatoxicosis, the renal clearance of the drug is apparently increased. On the basis of the present investigation, aflatoxin reduces the plasma concentration of CTC due to an increased elimination of the drug from the plasma. This increased elimination appears to be the result of a decreased binding of CTC to plasma proteins, thus allowing more unbound CTC to be available for elimination from the plasma by a means other than the liver, most likely via the kidney. REFERENCES
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Arakawa, A., and O. Ohe, 1975. Reduction of Clostriddium perfringes by feed additive antibiotics in the ceca of chickens infected with Eimeria tenella. Poultry Sci. 54:1000-1007. Brown, J.M.M., and L. Abrams, 1965. Biochemical studies on aflatoxicosis. Onderstepoort J. Vet. Res. 32:119-146. Bruning, J. L., and B. L. Kintz, 1968. Computational Handbook of Statistics. Scott Foresman Co., Glenview, IL. Craig, W. A., and B. Suh, 1980. Protein binding and the antibacterial effects. Methods for the determination of protein binding, Pages 265—297 in Antibiotics in Laboratory Medicine. Victor Lorian, ed. Williams and Wilkins Co., Baltimore, MD. Doumas, B. T., W. A. Watson, and H. G. Briggs, 1971. Albumin standards and the measurement of serum albumin with bromcresol green. Clin. Chim. Acta 31:87-96. Giambrone, J. J., D. L. Ewert, R. D. Wyatt, and C. S. Eidson, 1978. Effect of aflatoxin on the humoral and cell-mediated immune systems of the chicken. Am. J. Vet. Res. 39:305-308. Hamilton, P. B., and J. R. Harris, 1971. Interaction of aflatoxicosis with Candida albicans infections and other stresses in chickens. Poultry Sci. 50: 906-912. Hamilton, P. B., H.-T. Tung, R. D. Wyatt, and W. E. Donaldson, 1974. Interaction of dietary aflatoxin with some vitamin deficiencies. Poultry Sci. 53:871-877. Kohn, K. W., 1961. Determination of tetracyclines by extraction of fluorescent complexes. Anal. Chem. 33:862-866. Kunin, C. M., A. C. Dornbush, and M. Finland, 1959. Distribution and excretion of four tetracycline analogues in normal young men. J. Clin. Invest. 38:1950-1963. Levy, G., and A. Yacobi, 1974. Effect of plasma protein binding on elimination of warfarin. J. Pharm. Sci. 63:805-806. Lynd, J. Q., and F. T. Lynd, 1971. Mucor divergence in aflatoxin effects with duckling bile and plasma. Environ. Res. 4 : 3 1 6 - 3 2 4 . Nabney, J., and B. F. Nesbitt, 1965. A spectrophotometric method of determining the aflatoxins. Analyst 90:155-160. Nivas, S. C , M. D. York, and B. S. Pomeroy, 1976. Effects of different levels of chlortetracycline in
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