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Neurotoxicity of Triorthotolyl Phosphate in Chickens of Different Genotypes in the Presence and Absence of Deoxycorticosterone
Neurotoxicity of Triorthotolyl Phosphate in Chickens of Different Genotypes in the Presence and Absence of Deoxycorticosterone M. EHRICH,1'2 R. W. BRILES, 3 W. E. BRILES,3 E. A. DUNNINGTON,4 A. MARTIN,4 P. B. SIEGEL, 4 andW. B. GROSS 1 Virginia-Maryland Regional College of Veterinary Medicine, Blacksburg, Virginia 24061 and Department of Poultry Science, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061 and Department of Biological Sciences, Northern Illinois University, Dekalb, Illinois 60115
ABSTRACT Indices of acute and delayed toxicity following administration of triorthotolyl phosphate (TOTP) were measured in roosters from lines of chickens originating from the Cornell randombred population. Matings were designed to produce individuals that had presence or absence of allele 21 of the B blood system. Non-B21 individuals had allele 13 or 31. Acute inhibition of esterases (neurotoxic esterase, liver cholinesterase, plasma cholinesterases, and plasma carboxylesterases) occurred in all birds within 24 hr of a single oral dose of 360 mg/kg TOTP. Clinical signs of delayed neuropathy were evident within 12 days of TOTP administration, with no significant difference between genotypes. Dietary deoxycorticosterone (40 to 200 ppm) appeared incapable of statistically significant modification of the strong effects of TOTP. Activities of blood esterases were different between roosters having B 2 1 /B 2 1 and those with B 1 3 and/or B 3 1 . (Key words: chicken, triorthotolyl phosphate, deoxycorticosterone) 1986 Poultry Science 6 5 : 3 7 5 - 3 7 9 INTRODUCTION Response of chickens to infectious challenges can be modified by a variety of factors, including genetically d e t e r m i n e d systems (Gavora and Spencer, 1 9 7 8 , 1 9 8 3 ; Gavora, 1 9 8 4 ) , e n v i r o n m e n t (Gross and Siegel, 1 9 8 2 ; Gross, 1 9 8 4 ) , and administration of steroid h o r m o n e s such as corticosterone and d e o x y c o r t i c o s t e r o n e (DOC) (Gross et al, 1980a; Gross and Siegel, 1 9 8 1 ) . In a d d i t i o n t o protective effects described in these studies, D O C , b u t n o t cortic o s t e r o n e , was shown to have a beneficial effect o n feed efficiency. A l t h o u g h responses of chickens t o noninfectious challenges have been less extensively studied, neuropathological effects of t r i o r t h o t o l y l p h o s p h a t e (TOTP)
1
Virginia-Maryland Regional College of Veterinary Medicine. 2 To whom correspondence should be addressed. 3 Department of Biological Sciences, Northern Illinois University. 'Department of Poultry Science, Virginia Polytechnic Institute.
can be modified b y social stress (Ehrich and Gross, 1983) and administration of relatively low dietary c o n c e n t r a t i o n s of c o r t i c o s t e r o n e (Ehrich and Gross, 1 9 8 2 ) . Until t h e present s t u d y , effects of DOC against o r g a n o p h o s p h a t e poisoning had n o t been described. A gasoline additive and industrial chemical, T O T P is chemically and biologically related t o o r g a n o p h o s p h a t e s used as pesticides. As an o r g a n o p h o s p h a t e , it is capable of causing inhibition of activities of b l o o d and tissue cholinesterase e n z y m e s within m i n u t e s t o h o u r s after animals are exposed. In a d d i t i o n t o a c u t e esterase inhibition, T O T P can also i n d u c e a delayed n e u r o p a t h y , which is manifested b y distal axonal alteration primarily affecting t h e longest and largest nerve fibers. This n e u r o p a t h y is irreversible and progresses in a centripetal fashion, appearing days t o weeks after chickens are exposed t o T O T P (Davis and R i c h a r d s o n , 1980; Murphy, 1980). Several neurological m u t a n t s have been described for t h e fowl (Siegel, 1 9 7 9 ) , however, i n f o r m a t i o n on t h e influence of g e n o t y p e in response t o neurological toxins is lacking. T h e
375
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(Received for publication March 4, 1985)
376
EHRICH ET AL.
present study was designed to investigate this, using TOTP as a neurotoxin constituting a noninfectious challenge, and to ascertain modifying effects of dietary DOC. MATERIALS AND METHODS
Clinical Signs of Delayed Neuropathy. All live birds were observed daily for clinical signs and blindly scored for delayed neuropathy (Sprague et al, 1980; Ehrich and Gross, 1983). Scores were: 0, no neurological deficit; 1, altered gait; 2, some difficulty standing as well as walking; 3, great difficulty standing and walking; 4, prostrate and unwilling to stand; and 5, prostrate with both leg and wing involvement. Statistical Analyses. Analyses of variance were conducted for the variables measured in this experiment, estimating variation due to genotype, treatment, and a genotype by treatment interaction. Of the three blood esterases measured, a significant interaction occurred only in blood carboxylesterase which was further examined by additional analyses of variance within each genotype. RESULTS AND DISCUSSION
All roosters receiving the organophosphate TOTP exhibited acute and delayed effects, regardless of alleles at the B locus. Reactions were so strong that DOC could not bring about statistically significant modification of esterase inhibition (an acute effect) or clinical signs of delayed neuropathy (Table 1). Deoxycorticosterone can have a protective capability in chickens exposed to infectious challenges (Gross and Siegel, 1981; Gross, 1984) and a derogatory effect on nerve function by depressing excitation and decreasing the amplitude of electrical potentials received by the central nervous system (Dubrovsky et al, 1982). Ascertaining whether beneficial or detrimental effects of DOC would prevail in TOTP-induced delayed neuropathy requires
Downloaded from http://ps.oxfordjournals.org/ at NERL on June 7, 2015
Populations and Husbandry. White Leghorn chickens from the Cornell University randombred population were bred selectively to develop lines differing in antibody response to sheep red blood cell (SRBC) antigen (Siegel and Gross, 1980). These lines not only differ in response to SRBC antigen (Ubosieta/., 1985a,b) but also in resistance to various infectious agents (Gross et al, 1980b) and production traits (Siegel et al., 1982). Allelic frequencies of chickens from these populations have been determined for eight alloantigen systems (Dunnington et al, 1984). For the present study, matings were made so that individuals would have one of the following combinations of haplotypes of the B system: B21 IB21, B12/B13, or B 1 3 / B 3 1 . Each of the haplotype designations, B 1 , B1 , or B , is a symbol for a particular set of alloantigens determined by a minimum of three separate, but very tightly linked, loci within the major histocompatibility complex of the chicken (Briles et al., 1983); however, each haplotype is assumed to be nonvariant during the course of this study and will be referred to as an "allele". Alloantigen typing was performed as previously described (Dunnington et al, 1984). Upon hatching, chicks were wingbanded and reared on litter floors until 18 weeks of age and then transferred to wire battery cages measuring 30 X 50 X 50 cm. Feed and water were provided ad libitum. Birds were 32 weeks of age when administered TOTP. Treatments. Ninety-eight percent pure TOTP (Eastman Chemicals, Rochester, NY) was dissolved in corn oil such that the 360 mg/kg dose was contained in 1.0 ml. This organophosphate was administered by oral gavage (po). Deoxycorticosterone, >99% pure (Sigma Chemical Co., St. Louis, MO), was added to a mash breeder diet (16% protein and 2,772 kcal/kg metabolizable energy) at concentrations of 40, 80, 120, and 200 ppm. Birds (8/dosage level) were placed on the DOC feed 24 hr before challenge with TOTP and remained on it for 7 days after the organophosphate challenge. Three control groups of roosters (no TOTP administration) were fed 0, 80, or 200 ppm of
DOC. Numbers of roosters per treatment subclass are provided in the tables of results. Enzyme Assays. Twenty-four hours after TOTP administration, half of the birds in each treatment group were bled from the brachial vein and then killed by cervical dislocation and exsanguinated. Brains and livers were immediately removed and frozen at —70 C until biochemical analyses could be conducted (within 10 days). Spectrophotometric methods were used for determination of neurotoxic esterase of whole brain (Sprague et al, 1981), brain, blood (plasma) and liver cholinesterases (Ellman et al., 1961; Bellino et al., 1978), and plasma carboxylesterase (Levine and Murphy, 1977).
NEUROTOXICITY IN CHICKENS
377
TABLE 1. Mean clinical scores for chickens receiving triorthotolyl phosphate (TOTP) 360 mg/kg oral gavage1 Genotype
Days2 (X)
(Range
(X)
(Range)
(X)
(Range)
B2X/B21
12 14 16
1.4 3.2 3.3
.7-2.3 2.4-4.3 2.3-4.3
.9 2.2 2.4
0 -1.7 1.7-2.3 1.7-3.3
1.3 2.3 2.7
0 -3.3 1.3-5.0 1.3-5.0
B13/-3
12 14 16
1.0 2.5 2.8
0 -2.0 1.7-4.0 2.0-4.0
2.0 3.3 3.3
.7-3.0 1.7-4.3 1.7-4.3
2.7 4.1 4.0
2.0-3.0 3.3-4.0 3.0-5.0
DOC ,
o
DOC, 80 ppm
DOC, 200 ppm
1 Means of four birds on control diet, three birds on deoxycorticosterone (DOC) diet. Score of 0 = no neurological deficit, 1 = altered gait, 2 = moderate ataxia, 3 = severe ataxia, 4 = prostrate and unwilling to stand, and 5 = most severe neurological impairment, with both leg and wing involvement.
Days after triothotolyl phosphate administration.
3
B 1 3 / - = B 1 3 /B 1 3 or B 1 3 /B 3 1 .
further investigation and may, as our data suggest, vary depending on dosage of organophosphate and the presence of particular alleles for the B alloantigen system. Examination of acute neurotoxic effects of TOTP indicated that DOC had no significant effect on organophosphate-induced esterase inhibition in brain, plasma, or liver. For example, brain neurotoxic esterase, whose in-
hibition indicates potential for development of delayed neuropathy (Davis and Richardson, 1980), was less than 20% of control in all roosters given TOTP, and subsequently, clinical symptoms were observed in the birds. An organophosphate lacking capability to decrease brain acetylcholinesterase activity, TOTP caused no signs of acute cholinergic poisoning in roosters used in this experiment.
TABLE 2. Means ± standard errors of plasma esterase activity in samples from chickens with B 21 /B21 or B 1 3 /—' genotypes; comparison of treatments Esterase activity Treatment
n3
Acetylcholinesterase
Butyrylcholinesterase
Control DOC 80 DOC 200 TOTP
7 7 5 27 4
1.12 ± .05"y I.OI ± .osy 1.13 ± .07" .31 ± .022
.64 + .04" .47 ± .03y ( 73) .66 ± .07" (103) .24+ . 0 1 z ( 38)
Treatment
n
B21/B21
Control DOC 80 DOC 200 TOTP
4 3 2 14 4
( 90) (101) ( 28) Carboxylesterase
.48 .47 .94 .27
± + ± ±
.06y .12y .02" .02 z
( 98) (196) ( 56)
n
B13/-
3 4 3 13
.39 .49 .44 .23
± .01" ± .07" (126) ± .04" (113) + .02y ( 59)
x,y z
' Indicate differences (P<.05) among means within a column.
' B 1 3 / - = B I 3 / B 1 3 or B 1 3 /B 3 1 . 2 Micromol hydrolyzed/min/ml; values for genotypes combined for acetylcholinesterase and butyrylcholinesterase activities; ( ) = % of control. 3
n = Number of observations per mean.
"All groups of triorthotolyl phosphate-treated birds combined.
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2
378
EHRICH ET AL. TABLE 3. Means1 ± standard errors of plasma esterase activity in samples from chickens with or B13/—2 genotypes
Esterase
n3
B*1/B*1
Acetylcholinesterase Butyrylcholinesterase Carboxylesterase
23 22 23
.64 ± .08 .40 ± .04 .39±.05
+4 *"
B^'/B21
n3
B13/-
23 22 23
.61 ± .09 .36 + .04 .33±.03
1
Means include both triorthotolyl phosphate-treated and untreated birds in micromol hydrolyzed/min/ml.
2
B I 3 / - = B 1 3 /B 1 3 or B I 3 / B 3 1 .
3
n = Number of observations per mean.
4
+ (P<.07), *(P<.05) indicate differences between means in a row.
filtration of peripheral nerves (Calnek and Witter, 1978). Our results raise several questions, including the association of the B21 allele with resistance to a noninfectious neural disease, involvement of other alleles and loci with organophosphate effects, the role of antibody response in resistance to noninfectious challenges, and interactions of genetics with resistances to a combination of infectious and noninfectious challenges.
ACKNOWLEDGMENTS
Funds for this study were provided by Hatch projects of the Virginia Agricultural Experiment Station. Technical support was provided by Linda Correll and Constance Carr. Alloantigen determinations were supported in part by PHS grant CA 12796 to Northern Illinois University by the National Cancer Institute. REFERENCES Bellino, M., M. Ficarra, N. Frontali, F. Ghezzo, A. M. Guarcini, F. Orecchio, L. A. Serpietri, and M. E. Traina, 1978. A quick and simple method for the routine determination of acetyl- and butyrylcholinesterase in blood. Br. J. Ind. Med. 35: 161-167. Briles, W. E„ R. W. Briles, R. E. Taffs, and H. A. Stone, 1983. Resistance to a malignant lymphoma in chickens is mapped to subregion of major histocompatibility (B) complex. Science 219: 977-979. Briles, W. E., H. A. Stone, and R. K. Cole, 1977. Marek's disease: effects of B histocompatibility alloalleles in resistant and susceptible chicken lines. Science 195:193-195. Calnek, B. W., and R. L. Witter, 1978. Marek's disease. Page 385 in Diseases of Poultry. 7th ed. M. S. Hofstad, ed. The Iowa State University Press, Ames, IA. Davis, C. S., and R. J. Richardson, 1980. Organo-
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Extraneural esterases were, however, inhibited by TOTP. Although esterases outside the brain of chickens do not directly function as biochemical markers for acute or delayed toxicity induced by organophosphates (Pickering and Pickering, 1977), they do provide sites with which organophosphates interact. By providing such sites, these extraneural enzymes reduce the quantity of organophosphate available for inhibition of the esterases of the nervous system (Murphy, 1980). Of the plasma esterases examined, carboxylesterase activity was proportionally less sensitive to inhibition after administration of TOTP (Table 2), and alleles at the B alloantigen system had greater influence on this enzyme than on the others tested (Tables 2 and 3). As carboxylesterase activity was lower in non-B 21 birds, they may be more susceptible than B 2 1 birds to interactions involving organophosphates. Lower levels of these esterases after a first exposure to an organophosphate would decrease sites available outside the nervous system for organophosphate interaction after a second exposure. Although the presence of the B 21 allele did not protect roosters from acute or delayed neurotoxicity after a single high dose of TOTP, genotypic differences in plasma carboxylesterase inhibition were significant (P<.02) and approached significance (P<.07) for plasma butyrylcholinesterase (Table 3). It is, therefore, possible that chickens with the B21 allele could be less susceptible to neurotoxicity resulting from administration of organophosphate combinations or less overwhelming doses of TOTP. It has been reported previously that high resistance to marek's disease is associated with the B21 allele (Briles et al, 1977). Marek's disease, a herpes virus-induced lymphoma, is mainly characterized by a mononuclear in-
NEUROTOXICITY IN CHICKENS
Gross, W. B., P. B. Siegel, R. W. Hall, C. H. Domermuth, and R. T. Dubose, 1980b. Production and persistence of antibodies to sheep erythrocytes. 2. Resistance to infectious diseases. Poultry Sci. 59:205-214. Levine, B. S., and S. D. Murphy, 1977. Esterase inhibition and reactivation in relation to piperonyl butoxide-phosphorothionate interactions. Toxicol. Appl. Pharmacol. 4 0 : 3 7 9 - 3 9 1 . Murphy, S. D., 1980. Pesticides. Pages 357-408 in Casaratt and Doull's Toxicology, the Basic Science of Poisons. J. Doull, C. D. Klaassen, and M. O. Amdur, ed. 2nd ed. Macmillan, New York, NY. Pickering C. E., and R. G. Pickering, 1977. The interference by erythrocyte "acetylthiocholinesterase" in the estimation of the blood cholinesterase activity of the chicken. Toxicol. Appl. Pharmacol. 39:229-237. Siegel, P. B., 1979. Behavior genetics in chickens: A review. World's Poult. Sci. J. 35:9-19. Siegel, P. B., and W. B. Gross, 1980. Production and persistance of antibodies to sheep erythrocytes. I. Directional selection. Poultry Sci. 59:1—5. Siegel, P. B., W. B. Gross, and J. A. Cherry, 1982. Correlated responses of chickens to selection for production of antibodies to sheep erythrocytes. Anim. Blood Groups Biochem. Genet. 13: 291-297. Sprague, G. D., L. I. Sandvik, A. A. Bickford, and T. R. Castles, 1980. Evaluation of a sensitive grading system for assessing acute and subchronic delayed neurotoxicity in hens. Life Sci. 27:2523— 2528. Sprague, G. D., L. L. Sandvik, M. J. Brookins-Hendricks, and A. A. Bickford, 1981. Neurotoxicity of two organophosphorus ester flame retardants in hens. J. Toxicol. Environ. Health 8:507-518. Ubosi, C. O., E. A. Dunnington, W. B. Gross, and P. B. Siegel, 1985a. Divergent selection of chickens for antibody response to sheep erythrocytes: Kinetics of primary and secondary immunizations. Avian Dis. 29:347-355. Ubosi, C. O., W. B. Gross, and P. B. Siegel, 1985b. Divergent selection for antibody production to sheep erythrocytes: Age effect in parental lines and their crosses. Avian Dis. 29:150—158.
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phosphorus compounds. Pages 527—544 in Experimental and Clinical Neurotoxicology. P. S. Spencer and H. H. Schaumberg, ed. Williams & Wilkins, Baltimore, MD. Dubrovsky, B., D. Williams, and I. Kraulis, 1982. Effects of deoxycorticosterone and its ring A-reduced derivatives on the nervous system. Exp. Neurol. 78:728-739. Dunnington, E. A., R. W. Briles, W. E. Briles, W. B. Gross, and P. B. Siegel, 1984. Allelic frequencies in eight alloantigen systems of chickens selected for high and low antibody response to sheep red blood cells. Poultry Sci. 6 3 : 1 4 7 0 - 1472. Ehrich, M., and W. B. Gross, 1982. Effect of corticosterone on toxicity of malathion and TOTP in hens. Neurobehav. Toxicol. Teratol. 4:789-792. Ehrich, M., and W. B. Gross, 1983. Modification of triorthotolyl phosphate toxicity in chickens by stress. Toxicol. Appl. Pharmacol. 70:249-254. Ellman, G. L., K. D. Courtney, V. Andres Jr., and R. M. Featherstone, 1961. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol. 7:88—95. Gavora, J. S., 1984. Importance of genetic aspects of health in poultry production. Pages 31—35 in Proc. XVII World's Poultry Cong. Gavora, J. S., and J. L. Spencer, 1978. Breeding for genetic resistance to disease: Specific or general? World's Poult. Sci. J. 34:137-148. Gavora, J. S., and J. L. Spencer, 1983. Breeding for immune responsiveness and disease resistance. Anim. Blood Groups Biochem. Genet. 14: 159-180. Gross, W. B., 1984. Combined effects of deoxycorticosterone and furaltadone on Escherichia coli infection in chickens. Am. J. Vet. Res. 45:963-966. Gross, W. B., and P. B. Siegel, 1981. Some effects of feeding deoxycorticosterone to chickens. Poultry Sci. 6 0 : 2 2 3 2 - 2 2 3 9 . Gross, W. B., and P. B. Siegel, 1982. Socialization as a factor in resistance to infection, feed efficiency, and response to antigen in chickens. Am. J. Vet. Res. 43:2010-2012. Gross, W. B., P. B. Siegel, and R. T. DuBose, 1980a. Some effects of feeding corticosterone to chickens. Poultry Sci. 59:516-522.
379
ABSTRACT Indices of acute and delayed toxicity following administration of triorthotolyl phosphate (TOTP) were measured in roosters from lines of chickens originating from the Cornell randombred population. Matings were designed to produce individuals that had presence or absence of allele 21 of the B blood system. Non-B21 individuals had allele 13 or 31. Acute inhibition of esterases (neurotoxic esterase, liver cholinesterase, plasma cholinesterases, and plasma carboxylesterases) occurred in all birds within 24 hr of a single oral dose of 360 mg/kg TOTP. Clinical signs of delayed neuropathy were evident within 12 days of TOTP administration, with no significant difference between genotypes. Dietary deoxycorticosterone (40 to 200 ppm) appeared incapable of statistically significant modification of the strong effects of TOTP. Activities of blood esterases were different between roosters having B 2 1 /B 2 1 and those with B 1 3 and/or B 3 1 . (Key words: chicken, triorthotolyl phosphate, deoxycorticosterone) 1986 Poultry Science 6 5 : 3 7 5 - 3 7 9 INTRODUCTION Response of chickens to infectious challenges can be modified by a variety of factors, including genetically d e t e r m i n e d systems (Gavora and Spencer, 1 9 7 8 , 1 9 8 3 ; Gavora, 1 9 8 4 ) , e n v i r o n m e n t (Gross and Siegel, 1 9 8 2 ; Gross, 1 9 8 4 ) , and administration of steroid h o r m o n e s such as corticosterone and d e o x y c o r t i c o s t e r o n e (DOC) (Gross et al, 1980a; Gross and Siegel, 1 9 8 1 ) . In a d d i t i o n t o protective effects described in these studies, D O C , b u t n o t cortic o s t e r o n e , was shown to have a beneficial effect o n feed efficiency. A l t h o u g h responses of chickens t o noninfectious challenges have been less extensively studied, neuropathological effects of t r i o r t h o t o l y l p h o s p h a t e (TOTP)
1
Virginia-Maryland Regional College of Veterinary Medicine. 2 To whom correspondence should be addressed. 3 Department of Biological Sciences, Northern Illinois University. 'Department of Poultry Science, Virginia Polytechnic Institute.
can be modified b y social stress (Ehrich and Gross, 1983) and administration of relatively low dietary c o n c e n t r a t i o n s of c o r t i c o s t e r o n e (Ehrich and Gross, 1 9 8 2 ) . Until t h e present s t u d y , effects of DOC against o r g a n o p h o s p h a t e poisoning had n o t been described. A gasoline additive and industrial chemical, T O T P is chemically and biologically related t o o r g a n o p h o s p h a t e s used as pesticides. As an o r g a n o p h o s p h a t e , it is capable of causing inhibition of activities of b l o o d and tissue cholinesterase e n z y m e s within m i n u t e s t o h o u r s after animals are exposed. In a d d i t i o n t o a c u t e esterase inhibition, T O T P can also i n d u c e a delayed n e u r o p a t h y , which is manifested b y distal axonal alteration primarily affecting t h e longest and largest nerve fibers. This n e u r o p a t h y is irreversible and progresses in a centripetal fashion, appearing days t o weeks after chickens are exposed t o T O T P (Davis and R i c h a r d s o n , 1980; Murphy, 1980). Several neurological m u t a n t s have been described for t h e fowl (Siegel, 1 9 7 9 ) , however, i n f o r m a t i o n on t h e influence of g e n o t y p e in response t o neurological toxins is lacking. T h e
375
Downloaded from http://ps.oxfordjournals.org/ at NERL on June 7, 2015
(Received for publication March 4, 1985)
376
EHRICH ET AL.
present study was designed to investigate this, using TOTP as a neurotoxin constituting a noninfectious challenge, and to ascertain modifying effects of dietary DOC. MATERIALS AND METHODS
Clinical Signs of Delayed Neuropathy. All live birds were observed daily for clinical signs and blindly scored for delayed neuropathy (Sprague et al, 1980; Ehrich and Gross, 1983). Scores were: 0, no neurological deficit; 1, altered gait; 2, some difficulty standing as well as walking; 3, great difficulty standing and walking; 4, prostrate and unwilling to stand; and 5, prostrate with both leg and wing involvement. Statistical Analyses. Analyses of variance were conducted for the variables measured in this experiment, estimating variation due to genotype, treatment, and a genotype by treatment interaction. Of the three blood esterases measured, a significant interaction occurred only in blood carboxylesterase which was further examined by additional analyses of variance within each genotype. RESULTS AND DISCUSSION
All roosters receiving the organophosphate TOTP exhibited acute and delayed effects, regardless of alleles at the B locus. Reactions were so strong that DOC could not bring about statistically significant modification of esterase inhibition (an acute effect) or clinical signs of delayed neuropathy (Table 1). Deoxycorticosterone can have a protective capability in chickens exposed to infectious challenges (Gross and Siegel, 1981; Gross, 1984) and a derogatory effect on nerve function by depressing excitation and decreasing the amplitude of electrical potentials received by the central nervous system (Dubrovsky et al, 1982). Ascertaining whether beneficial or detrimental effects of DOC would prevail in TOTP-induced delayed neuropathy requires
Downloaded from http://ps.oxfordjournals.org/ at NERL on June 7, 2015
Populations and Husbandry. White Leghorn chickens from the Cornell University randombred population were bred selectively to develop lines differing in antibody response to sheep red blood cell (SRBC) antigen (Siegel and Gross, 1980). These lines not only differ in response to SRBC antigen (Ubosieta/., 1985a,b) but also in resistance to various infectious agents (Gross et al, 1980b) and production traits (Siegel et al., 1982). Allelic frequencies of chickens from these populations have been determined for eight alloantigen systems (Dunnington et al, 1984). For the present study, matings were made so that individuals would have one of the following combinations of haplotypes of the B system: B21 IB21, B12/B13, or B 1 3 / B 3 1 . Each of the haplotype designations, B 1 , B1 , or B , is a symbol for a particular set of alloantigens determined by a minimum of three separate, but very tightly linked, loci within the major histocompatibility complex of the chicken (Briles et al., 1983); however, each haplotype is assumed to be nonvariant during the course of this study and will be referred to as an "allele". Alloantigen typing was performed as previously described (Dunnington et al, 1984). Upon hatching, chicks were wingbanded and reared on litter floors until 18 weeks of age and then transferred to wire battery cages measuring 30 X 50 X 50 cm. Feed and water were provided ad libitum. Birds were 32 weeks of age when administered TOTP. Treatments. Ninety-eight percent pure TOTP (Eastman Chemicals, Rochester, NY) was dissolved in corn oil such that the 360 mg/kg dose was contained in 1.0 ml. This organophosphate was administered by oral gavage (po). Deoxycorticosterone, >99% pure (Sigma Chemical Co., St. Louis, MO), was added to a mash breeder diet (16% protein and 2,772 kcal/kg metabolizable energy) at concentrations of 40, 80, 120, and 200 ppm. Birds (8/dosage level) were placed on the DOC feed 24 hr before challenge with TOTP and remained on it for 7 days after the organophosphate challenge. Three control groups of roosters (no TOTP administration) were fed 0, 80, or 200 ppm of
DOC. Numbers of roosters per treatment subclass are provided in the tables of results. Enzyme Assays. Twenty-four hours after TOTP administration, half of the birds in each treatment group were bled from the brachial vein and then killed by cervical dislocation and exsanguinated. Brains and livers were immediately removed and frozen at —70 C until biochemical analyses could be conducted (within 10 days). Spectrophotometric methods were used for determination of neurotoxic esterase of whole brain (Sprague et al, 1981), brain, blood (plasma) and liver cholinesterases (Ellman et al., 1961; Bellino et al., 1978), and plasma carboxylesterase (Levine and Murphy, 1977).
NEUROTOXICITY IN CHICKENS
377
TABLE 1. Mean clinical scores for chickens receiving triorthotolyl phosphate (TOTP) 360 mg/kg oral gavage1 Genotype
Days2 (X)
(Range
(X)
(Range)
(X)
(Range)
B2X/B21
12 14 16
1.4 3.2 3.3
.7-2.3 2.4-4.3 2.3-4.3
.9 2.2 2.4
0 -1.7 1.7-2.3 1.7-3.3
1.3 2.3 2.7
0 -3.3 1.3-5.0 1.3-5.0
B13/-3
12 14 16
1.0 2.5 2.8
0 -2.0 1.7-4.0 2.0-4.0
2.0 3.3 3.3
.7-3.0 1.7-4.3 1.7-4.3
2.7 4.1 4.0
2.0-3.0 3.3-4.0 3.0-5.0
DOC ,
o
DOC, 80 ppm
DOC, 200 ppm
1 Means of four birds on control diet, three birds on deoxycorticosterone (DOC) diet. Score of 0 = no neurological deficit, 1 = altered gait, 2 = moderate ataxia, 3 = severe ataxia, 4 = prostrate and unwilling to stand, and 5 = most severe neurological impairment, with both leg and wing involvement.
Days after triothotolyl phosphate administration.
3
B 1 3 / - = B 1 3 /B 1 3 or B 1 3 /B 3 1 .
further investigation and may, as our data suggest, vary depending on dosage of organophosphate and the presence of particular alleles for the B alloantigen system. Examination of acute neurotoxic effects of TOTP indicated that DOC had no significant effect on organophosphate-induced esterase inhibition in brain, plasma, or liver. For example, brain neurotoxic esterase, whose in-
hibition indicates potential for development of delayed neuropathy (Davis and Richardson, 1980), was less than 20% of control in all roosters given TOTP, and subsequently, clinical symptoms were observed in the birds. An organophosphate lacking capability to decrease brain acetylcholinesterase activity, TOTP caused no signs of acute cholinergic poisoning in roosters used in this experiment.
TABLE 2. Means ± standard errors of plasma esterase activity in samples from chickens with B 21 /B21 or B 1 3 /—' genotypes; comparison of treatments Esterase activity Treatment
n3
Acetylcholinesterase
Butyrylcholinesterase
Control DOC 80 DOC 200 TOTP
7 7 5 27 4
1.12 ± .05"y I.OI ± .osy 1.13 ± .07" .31 ± .022
.64 + .04" .47 ± .03y ( 73) .66 ± .07" (103) .24+ . 0 1 z ( 38)
Treatment
n
B21/B21
Control DOC 80 DOC 200 TOTP
4 3 2 14 4
( 90) (101) ( 28) Carboxylesterase
.48 .47 .94 .27
± + ± ±
.06y .12y .02" .02 z
( 98) (196) ( 56)
n
B13/-
3 4 3 13
.39 .49 .44 .23
± .01" ± .07" (126) ± .04" (113) + .02y ( 59)
x,y z
' Indicate differences (P<.05) among means within a column.
' B 1 3 / - = B I 3 / B 1 3 or B 1 3 /B 3 1 . 2 Micromol hydrolyzed/min/ml; values for genotypes combined for acetylcholinesterase and butyrylcholinesterase activities; ( ) = % of control. 3
n = Number of observations per mean.
"All groups of triorthotolyl phosphate-treated birds combined.
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2
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EHRICH ET AL. TABLE 3. Means1 ± standard errors of plasma esterase activity in samples from chickens with or B13/—2 genotypes
Esterase
n3
B*1/B*1
Acetylcholinesterase Butyrylcholinesterase Carboxylesterase
23 22 23
.64 ± .08 .40 ± .04 .39±.05
+4 *"
B^'/B21
n3
B13/-
23 22 23
.61 ± .09 .36 + .04 .33±.03
1
Means include both triorthotolyl phosphate-treated and untreated birds in micromol hydrolyzed/min/ml.
2
B I 3 / - = B 1 3 /B 1 3 or B I 3 / B 3 1 .
3
n = Number of observations per mean.
4
+ (P<.07), *(P<.05) indicate differences between means in a row.
filtration of peripheral nerves (Calnek and Witter, 1978). Our results raise several questions, including the association of the B21 allele with resistance to a noninfectious neural disease, involvement of other alleles and loci with organophosphate effects, the role of antibody response in resistance to noninfectious challenges, and interactions of genetics with resistances to a combination of infectious and noninfectious challenges.
ACKNOWLEDGMENTS
Funds for this study were provided by Hatch projects of the Virginia Agricultural Experiment Station. Technical support was provided by Linda Correll and Constance Carr. Alloantigen determinations were supported in part by PHS grant CA 12796 to Northern Illinois University by the National Cancer Institute. REFERENCES Bellino, M., M. Ficarra, N. Frontali, F. Ghezzo, A. M. Guarcini, F. Orecchio, L. A. Serpietri, and M. E. Traina, 1978. A quick and simple method for the routine determination of acetyl- and butyrylcholinesterase in blood. Br. J. Ind. Med. 35: 161-167. Briles, W. E„ R. W. Briles, R. E. Taffs, and H. A. Stone, 1983. Resistance to a malignant lymphoma in chickens is mapped to subregion of major histocompatibility (B) complex. Science 219: 977-979. Briles, W. E., H. A. Stone, and R. K. Cole, 1977. Marek's disease: effects of B histocompatibility alloalleles in resistant and susceptible chicken lines. Science 195:193-195. Calnek, B. W., and R. L. Witter, 1978. Marek's disease. Page 385 in Diseases of Poultry. 7th ed. M. S. Hofstad, ed. The Iowa State University Press, Ames, IA. Davis, C. S., and R. J. Richardson, 1980. Organo-
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Extraneural esterases were, however, inhibited by TOTP. Although esterases outside the brain of chickens do not directly function as biochemical markers for acute or delayed toxicity induced by organophosphates (Pickering and Pickering, 1977), they do provide sites with which organophosphates interact. By providing such sites, these extraneural enzymes reduce the quantity of organophosphate available for inhibition of the esterases of the nervous system (Murphy, 1980). Of the plasma esterases examined, carboxylesterase activity was proportionally less sensitive to inhibition after administration of TOTP (Table 2), and alleles at the B alloantigen system had greater influence on this enzyme than on the others tested (Tables 2 and 3). As carboxylesterase activity was lower in non-B 21 birds, they may be more susceptible than B 2 1 birds to interactions involving organophosphates. Lower levels of these esterases after a first exposure to an organophosphate would decrease sites available outside the nervous system for organophosphate interaction after a second exposure. Although the presence of the B 21 allele did not protect roosters from acute or delayed neurotoxicity after a single high dose of TOTP, genotypic differences in plasma carboxylesterase inhibition were significant (P<.02) and approached significance (P<.07) for plasma butyrylcholinesterase (Table 3). It is, therefore, possible that chickens with the B21 allele could be less susceptible to neurotoxicity resulting from administration of organophosphate combinations or less overwhelming doses of TOTP. It has been reported previously that high resistance to marek's disease is associated with the B21 allele (Briles et al, 1977). Marek's disease, a herpes virus-induced lymphoma, is mainly characterized by a mononuclear in-
NEUROTOXICITY IN CHICKENS
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