Aflatoxicosis and Intrinsic Coagulation Function in Broiler Chickens ' 2

Transcription

1 Aflatoxicosis and Intrinsic Coagulation Function in Broiler Chickens ' 2 J. A. DOERR 4 and P. B. HAMILTON Departments of Poultry Science and Microbiology, North Carolina State University, Raleigh, North Carolina (Received for publication September 25, 1980) ABSTRACT Aflatoxin was fed (0,.625, 1.25, 2.5, 5.0, and 10.0 Mg/g) to broiler chickens from day-old to 3 weeks of age when the birds were bled and intrinsic coagulation parameters measured. Clotting times of whole blood were increased by aflatoxin (2.5, 5.0, and 10.0 Mg/g) and decreased in blood samples activated by contact with the surface of crushed glass. There was no interaction of aflatoxin with the contact phenomenon. Partial thromboplastin times were significantly (P<.05) prolonged by 5.0 and 10.0 Mg/g aflatoxin. Intrinsic activity as judged by whole blood thromboplastin generation was reduced nearly 40% by those two levels of aflatoxin. Activity analogous to human factor VIII was depressed by the two highest doses, but factor IX was significantly (P<.05) reduced by only the highest dose fed, 10 Mg/g. These data suggest that chickens possess an intrinsic coagulation mechanism that is sensitive to aflatoxin but that the factor or factors responsible for the contact response are refractory to this important mycotoxin. (Key words: blood, coagulation, broilers, intrinsic clotting pathway, thromboplastin, aflatoxicosis) INTRODUCTION Aflatoxin has many profound effects in chickens including abnormal blood clotting (Doerr et al, 1974, 1976). The coagulopathy associated with aflatoxicosis appeared to be primarily hypoprothombinemia, which is provoked even by concentrations of the mycotoxin too small to inhibit growth (Doerr et al., 1976). However, this assessment is based on evaluation of the extrinsic and common pathways only and without consideration of the intrinsic pathway. The existence and role of intrinsic coagulation in chickens has been questioned (Stopforth, 1970; Archer, 1971; Bigland and Triantaphyllopoulos, 1960), but our laboratory, using improved methodology, confirmed the existence of intrinsic acitivty in chicken blood (Doerr and Hamilton, 1981). It then became possible to investigate the response of intrinsic 1 Paper No of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, NC The use of trade names in this publication does not imply endorsement by the North Carolina Agricultural Research Service of the product named nor criticism of similar ones not mentioned. 3 A preliminary report of this paper was presented at the 66th Annual Meeting of the Poultry Science Association, Auburn, AL, "Present address: Department of Poultry Science, University of Maryland, College Park, MD Poultry Science 60: coagulation to aflatoxin. Blood clotting can be explained on the basis of a model that assumes a series of reactions initiated by factors internal or intrinsic to the blood and a series of reactions initiated by factors external or extrinsic to the blood system (Doerr et al., 1976; Sirridge, 1974). The later portions of the two series are the same or common. Thus, coagulation can be described as a composite of an intrinsic pathway, an extrinsic pathway, and a common pathway. The intrinsic system of humans that serves as a useful model for avian coagulation is initiated by a factor (XII) activated by contact with a hydrophilic surface. This activated factor in turn activates factor XI and the two form a complex that activates factor IX that, with factor VIII and platelet factor as cofactors, activates factor X of the common pathway as the terminal step of the intrinsic pathway. The purpose of the present investigation was to determine the response of intrinsic activity in chickens during aflatoxicosis and whether intrinsic factors were involved in the multifactorial coagulopathy caused by dietary aflatoxin in broiler chickens (Doerr et al, 1976). MATERIALS AND METHODS Husbandry. Day-old male broiler chicks (Hubbard X Hubbard) were obtained from the university flock. Chicks were reared in electri- 1406

2 AFLATOXIN AND HEMOSTASIS 1407 cally heated batteries under continuous illumination with water and diets provided ad libitum. The experimental design was completely randomized. There were 4 replicates of 10 birds at each treatment level. At three weeks of age, the experiments were terminated and coagulation variables measured. Aflatoxin Production. Aflatoxin was produced by growing Aspergillus parasiticus NRRL 2999 on rice by the method of Shotwell et al. (1966) as modified by West et al. (1973). The moldy rice was steamed, dried, and ground to a fine powder that was analyzed spectrophotometrically for its aflatoxin content by the method of Nabney and Nesbitt (1965) with the modification of Wiseman et al. (1970). Identification and confirmation of aflatoxin were by the chromatographic method of Pons et al. (1966). Weighed amounts of rice powder were added to a commercial broiler-starter ration free of any medications. At its highest concentration, the rice powder never exceeded 1% of the diet. The concentrations of aflatoxin were 0,.625, 1.25, 2.5, 5.0, and 10 jxglg of toxin in diet. Blood. Blood samples were obtained by cardiac puncture using disposable plastic syringes fitted with needles siliconized by treatment with Siliclad (Arthur H. Thomas Co., Philadelphia, PA). Siliconization of needles and glassware was necessary to reduce contact activation of the initiating factors of the intrinsic pathway. For analysis of contact factors, clotting time measurements began when one half of the total blood volume to be drawn had entered the syringe. The syringes were inverted to mix the blood. In other experiments blood was obtained as stated except that it was dispensed into siliconized tubes containing.18 M sodium citrate in the ratio of one part citrate solution to nine parts blood. Blood is treated with citrate to render unavailable Ca ++, which is required for coagulation. (Blood treated in such a fashion is termed anticoagulated blood.) Plasma was recovered following centrifugation of anticoagulated blood at 1,000 X g for 15 min at 4 C. Measurements on plasma were made within two hours of collection. Coagulation Tests. Whole blood clotting times were measured by splitting blood samples from each bird between two tubes, one of which contained approximately.3 ml acidcleaned, crushed glass while the other had no additive. The tubes were stoppered and simultaneously tilted at 15 sec intervals until a firm clot was formed. Partial thromboplastin times were measured according to Sirridge (1974) except that platelet factor substitute was prepared from chicken brains following the procedure of Bell and Alton (1954). Whole blood thromboplastin generation was as according to Sirridge (1974) except that the reagents were incubated for 15 min to insure maximal thromboplastin formation. This test measures the efficiency of formation of intrinsic thromboplastin by generating procoagulant activity in dilute whole blood. Lysed red blood cells provide platelet factors (this commonly accepted terminology does not imply that chicken blood has platelets) and dilution minimizes the effects of any trace tissue thromboplastin contamination. Whole blood thromboplastin generation mixtures were corrected for intrinsic factor deficiencies by adding either 1) factor VIII correction reagent (BaSCU adsorbed, citrated plasma), or 2) factor IX correction reagent (oxalated, aged serum), which also contained factors XI and XII but whose presence and possible contributions were controlled by the use of 3) factor XI and XII correction reagent (BaS04 adsorbed, oxalated, aged serum). The correction reagents were prepared according to Sirridge (1974). Statistics. Data were analyzed statistically by analysis of variance or by a paired t-test, as appropriate (Snedecor and Cochran, 1967). Data reported in percent were submitted to logarithmic transformation prior to analysis of variance. RESULTS The relationship of contact activation to dietary aflatoxin is shown in Figure 1. Aflatoxin produced greater than a twofold increase in clotting times of whole blood independent of activation by contact with crushed glass. The increase was significant (P<.05) at 2.5 Mg/g and above, which in this experimental system are growth inhibitory doses (Smith and Hamilton, 1970). Over the entire dose response curve, activated samples displayed a mean reduction of 84 sec in clotting time from the respective nonactivated control. The lack of interaction suggests that the contact phenomenon in chicken blood is insensitive to dietary aflatoxin. The results of partial thromboplastin time tests are given in Figure 2. This test measures the function of intrinsic and common pathway reactants while by-passing extrinsic factors.

3 1408 DOERR AND HAMILTON AFLATOXIN 5.0 1/j.g/g) FIG. 1. Effect of graded levels of dietary aflatoxin on nonactivated and glass activated whole blood clotting times. Each data point and vertical bar represents the mean ± SEM of 30 birds. Aflatoxin at 5.0 and 10.0 /ig/g significantly (P<.05) prolonged these times from the control value of 33 sec, which compares favorably with normal values for this test in humans (Holvey, 1972). However, this generalized test measures, in part, the function of the common clotting pathway that has been shown to be affected by aflatoxin (Doerr et al, 1976). Therefore, the clotting times developed by intrinsically generated thromboplastin were measured (Fig. 3) because they reflect activity of the intrinsic pathway alone (Sirridge, 1974). This clotting parameter also was significantly altered by 5.0 and 10.0 ng/g aflatoxin in diet. The mean prolongation of clotting time was 30 sec, which is equivalent to a reduction in intrinsic activity of 38%. Such a prolongation means that blood from birds fed aflatoxin are deficient in factor(s) of the intrinsic pathway. Figure 4 shows the results of adding reagents in specific factors in an attempt to correct and identify the deficiency(ies). The addition of contact factors XI and XII, which the model for intrinsic coagulation assumes to be initiators of the pathway, was without effect. This agrees with the aflatoxin insensitivity of contact activation seen in Figure 1. The addition of factor VIII or factor IX caused a significant shortening of the clotting times prolonged by aflatoxin. From these data (Fig. 4) it was possible to calculate the effect of aflatoxin on the two factors over the entire dose response curve. The calculations assumed each curve was 50 AFLATOXIN (/ig/g) FIG. 2. Effect of dietary aflatoxin on partial thromboplastin times. Each data point and vertical bar represents the mean ± SEM of 4 groups of 10 birds. FIG. 3. Effect of dietary aflatoxin on intrinsic thromboplastin activity that was assayed by measuring the clotting time of plasma after the addition of dilute whole chicken blood treated to allow the maximum generation of intrinsic thromboplastin. Each point and vertical bar represents the mean ± SEM of 4 groups of 10 birds.

4 AFLATOXIN AND HEMOSTASIS 1409 FACTOR EX AFLATOXIN (jig/g) FIG. 4. Effect of factor correction on intrinsic thromboplastin. Correction reagents were BaS0 4 adsorbed plasma (factor VIII). oxalated aged serum (factor IX), and adsorbed aged serum (factors XI, XII). Each point and vertical bar represents the mean + SEM of 4 groups of 10 birds. independent and the appropriate control (zero aflatoxin value) for a curve was that of the particular curve. Further, the failure of correction with a particular factor to restore clotting time to the zero aflatoxin value is assumed to be a deficiency in another factor (e.g., factor VIII addition at 10 fig/g aflatoxin gave only partial correction and the uncorrected portion is assumed to represent a deficiency in factor IX). These assumptions appear necessary and valid (Sirridge, 1974). The calculations were made according to the formula ^ LL] + l) x 100 = Activity where T 0 is the clotting time (sec) with zero aflatoxin and T t is the clotting time (sec) with a given amount of aflatoxin. If T 0 and T t are from the factor VIII curve, then the activity (%) applies to factor IX, and vice versa. Figure 5 shows that aflatoxin caused a significant (P<.05) decrease in factor VIII activity at doses of 5 and 10 /g/g- The decrease was about one-half normal values. Factor IX activity (Fig. 5) was reduced significantly (P<.05) only at 10 /ig/g. The reductions found in these two factors are generally commensurate with the reduction in intrinsic activity measured in the whole blood thromboplastin generation test (Fig. 3). > O < rr e < ACTOR Vm- AFLATOXIN (^g/g) FIG. 5. Effect of dietary aflatoxin on factors VIII and IX activity. Each point and vertical bar represents the mean ± SEM of 4 groups of 10 birds. DISCUSSION It seems clear from the present results that aflatoxin has a deleterious effect on the intrinsic coagulation system of chickens. The locus of its action does not appear to include the aspect of surface activation which presumably initiates the intrinsic hemostatic cascade. This is supported by the failure to demonstrate any effect of aflatoxin on contact response (Fig. 1) or to observe any correction when factor XI/XII reagent was added to whole blood thromboplastin generation mixtures (Fig. 4). Factors XI and XII are factors activated by contact (Sirridge, 1974) and presumably are initiating factors of intrinsic coagulation. Impairment of intrinsic function was indicated by the prolongation of partial prothrombin times (Fig. 2) and of whole blood thromboplastin generation (Fig. 3). Impairment of the latter measure was corrected by the addition of factors VIII or IX. Quantitation of this corrective effect revealed that factor VIII was decreased by about one-half at doses of 5 and 10 jug/g while factor IX was decreased significantly at only 10 Mg/g. The greater sensitivity of factor VIII to aflatoxin recalled the report (Barrow and Graham, 1974) of a possible role for albumin in the quaternary structure of factor VIII. Because albumin concentration is highly sensitive to aflatoxin (Tung et ah, 1975), an interference

5 1410 DOERR AND HAMILTON with production or activity of factor VIII during aflatoxicosis might be expected. Factor VIII alone of the clotting factors is not synthesized in the liver (Graham et al., 1951) but rather in the spleen where it is stored (Libre et al., 1968). Aflatoxin is not only a potent hepatotoxin but a splenotoxin as well (Smith and Hamilton, 1970). Thus, it appears that the primary site of aflatoxin inhibition of intrinsic coagulation in the chicken is a reduction of factor VIII activity and that the secondary site of inhibition is factor IX. The importance of this inhibition of intrinsic coagulation during aflatoxicosis is npt entirely clear. While decreased capacity would be deleterious when intrinsic coagulation reactions are necessary to hemostasis of the animal, the importance of intrinsic reactions to overall hemostatic competence, aside from their existence, is unknown. However, it is known that prothrombin, which is a component of the common clotting pathway, was reduced in concentration by a dose of.625 jug/g (Doerr et al., 1976), making it about ten times more sensitive than the intrinsic factor VIII. Thus, the coagulopathy of aflatoxicosis remains primarily a hypoprothrombinemia with the feature that every hemostatic component measured to date, except for contact factors (XI/XII), is reduced in concentration during the severe toxicosis (10 jug/g). A possibly important aspect of intrinsic coagulation not studied in this investigation was the response, if any, of thrombocytes. Platelets, the human equivalent of thrombocytes, liberate an essential component of the intrinsic system. Whether thrombocytes have such a role and whether it would be inhibited by aflatoxin is not known, but thrombocyte numbers are not altered during aflatoxicosis (R. D. Wyatt, personal communication) and their phagocytic function is refractory to aflatoxin (Chang and Hamilton, 1979). The data reported here affirm the presence of a functional intrinsic coagulation activity in chickens contrary to certain reports (Archer, 1971; Bigland and Triantaphyllopoulos, I960; Stopforth, 1970). We account for the difference by our use of divided samples to reduce variation and by the use of human models as analogies rather than homologies. While the description and purpose of the avian intrinsic coagulation pathway are not entirely clear, the system exists, and, furthermore, it is impaired during aflatoxicosis in chickens. ACKNOWLEDGMENTS We thank Nancy Bailey, Sharon West, and Gorum Whitaker for technical assistance. REFERENCES Archer, R. K., Blood coagulation. Pages in Physiology and biochemistry of the domestic fowl. D. J. Bell and B. M. Freeman, ed. Academic Press, New York, NY. Barrow, E. S., and J. B. Graham, Albumin and coagulation factor VIII (AHF). Amer. J. Physiol. 227: Bell, W. N., and H. G. Alton, A brain extract as a substitute for platelet suspensions in the thromboplastin generation test. Nature 174: Bigland, C. H., and D. C. Triantaphyllopoulos, A re-evaluation of the clotting time of chicken blood. Nature 186:644. Chang, C. F., and P. B. Hamilton, Refractory phagocytosis by chicken thrombocytes during aflatoxicosis. Poultry Sci. 58: Doerr, J. A., W. E. Huff, H. T. Tung, R. D. Wyatt, and P. B. Hamilton, A survey of T-2 toxin, ochratoxin, and aflatoxin for their effects on the coagulation of blood in young broiler chickens. Poultry Sci. 53: Doerr, J. A., R. D. Wyatt, and P. B. Hamilton, Impairment of coagulation function during aflatoxicosis in young chickens. Toxicol. Appl. Pharmacol. 35: Graham, J. B., D. L. Collins, I. D. Godwin, and K. M. Brinkhous, Antihemophilic factor: levels in canine and human plasmas, and following liver injury and Dicumarol. Fed. Proc. 10: Holvey, D. N., The Merck manual of diagnosis and therapy. Pages Merck, Sharp and Dohme Research Laboratories, Rahway, NJ. Libre, E. P., D. H. Cowan, S. P. Watkins, and N. P. Shulman, Relationships between spleen, platelets, and factor VII levels. Blood 31: Nabney, J., and B. F. Nesbitt, A spectrophotometric method of determining the aflatoxins. Analyst 90: Pons, W. A., A. F. Cucullu, L. S. Lee, J. A. Robertson, A. O. Franz, and L. A. Goldblatt, Determination of aflatoxins in agricultural products: use of aqueous acetone for extraction. J. Assoc. Offic. Agr. Chem. 49: Shotwell, O. L., C. W. Hesseltine, R. D. Stubblefield, andw. G.Sorenson, Production of aflatoxin on rice. Appl. Microbiol. 14: Sirridge, M. S., Laboratory evaluation of hemostasis. Lea & Febiger, Philadelphia, PA. Smith, J. W., and P. B. Hamilton, Aflatoxicosis in the broiler chicken. Poultry Sci. 49: Snedecor, G. W., and W. G. Cochran, Statistical methods. Iowa State University Press, Ames, IA. Stopforth, A., A study of coagulation mechanisms in domestic chickens. J. Comp. Pathol. 80: Tung, H. T., R. D. Wyatt, P. Thaxton, and P. B. Hamilton, Concentrations of serum proteins

6 AFLATOXIN AND HEMOSTASIS 1411 during aflatoxicosis. Toxicol. Appl. Pharmacol. 34: West, S., R. D. Wyatt, and P. B. Hamilton, Improved yield of aflatoxin by incremental increases of temperature. Appl. Microbiol. 25: Wiseman, H. G., W. C. Jacobson, and W. C. Harmeyer, Note on removal of pigments from chloroform extracts of aflatoxin cultures with copper carbonate. J. Assoc. Offic. Agr. Chem. 50: