Tryptophan 2,3-dioxygenase activity in turkey poults infected with Bordetella avium

Comp. Biochem. Physiol. Vol. 97B, No. 4, pp. 755-759, 1990 Printed in Great Britain

0305-0491/90 $3.00 + 0.00 © 1990 Pergamon Press plc

TRYPTOPHAN 2,3-DIOXYGENASE ACTIVITY IN TURKEY POULTS INFECTED WITH BORDETELLA A VIUM* ANDREW G. YERSIN,'~F. W. EDENS and D. G. SIMMONS~ North Carolina State University, Department of Poultry Science, Box 7635, Raleigh, NC 27695-7635, USA (Tel: 919 737 2649) (Recewed 11 June 1990)

Liver tryptophan 2,3-dioxygenase (TPO) activity was depressed significantly by the Bordetella avium infection localized in the trachea of the turkey poult. 2. Tryptophan, given orally, induced a significant increase in TPO activity in both control and infected poults. 3. Hydrocortisone induced TPO activity in the turkey in a dose dependent manner. Abstract--1.

INTRODUCTION Bordetella avium, formerly identified as Alcaligenes faecalis, is a gram negative, aerobic bacillus causing turkey rhinotracheitis or turkey coryza (Simmons et al., 1979). The disease is characterized clinically by excessive mouth breathing, rhinitis, foamy conjunctivitis, and loss of the ciliated epithelium lining the trachea (Saif et al., 1980; Simmons et aL, 1980a). Numerous physiological dysfunctions, associated with the disease and indicative of a stress response in the turkey poult, have been reported and include an impaired cellular immune system (Simmons et al., 1980b), a decrease in body weight gain (Edens et al., 1984; Simmons et al., 1979; Simmons et al., 1980a), an increase in serum corticosterone (McCorkle et al., 1985), variation in the normal body temperature (Edens et al., 1984), and alteration in the concentration of whole brain monoamines (Edens et al., 1987). In addition, B. avium was shown to produce a variety of toxins, including a heat-labile toxin (Rimler, 1985), a heat-stable toxin (Simmons et al., 1986), a tracheal cytotoxin (Gentry-Weeks et al., 1988) and an endotoxin (Edens et al., 1984; McCorkle et al., 1985; Simmons et al., 1986), all of which are considered to be stressors. Tryptophan 2,3-dioxygenase or tryptophan pyrrolase (TPO) catalyzes the oxidation of tryptophan to formylkynurenine in the liver (Feigelson and Greengard, 1961; Badawy and Evans, 1975). Formylkynurenine then enters the kynurenine pathway and ultimately is metabolized to either xanthurenic acid and excreted in the urine or to NAD, CO2 and H20 (Kim and Hill, 1967; Takikawa et aL, 1986). The metabolism of tryptophan to nicotinamide and its incorporation into the di- and

*The use of tradenames in this publication does not imply endorsement of the product named nor criticism of similar products not mentioned. tCurrent address: USDA-ARS, Veterinary Toxicology and Entomology Research Laboratory, PO Drawer GE, College Station, TX 77841, USA. :~Current address: Pennsylvania State University, 115 W. L. Henning Building, University Park, PA 16802, USA.

triphospho-pyridine nucleotides (DPN, TPN) establishes the pathway to be of major significance for exothermic reactions. In addition, the fate of tryptophan is also influenced by the metabolic pathway which results in the synthesis of serotonin. The biosynthesis of serotonin involves the 5-hydroxylation of its precursor amino acid, L-tryptophan, and is influenced by the availability of L-tryptophan (Fernstrom and Wurtman, 1971). TPO, therefore, is of interest in bordetellosis, since serotonin is depressed by the disease state (Edens et al., 1987) and is a pivotal enzyme in tryptophan metabolism. It was shown that both corticosteroids (Civen and Knox, 1959; Kim and Hill, 1967; Knox and Auerbach, 1955; Moon, 1985; Peterkofsky, 1968; Renkawitz et al., 1984) and tryptophan loading (Civen and Knox, 1959; Greengard and Feigelson, 1961; Knox and Piras, 1966; Moon, 1985) induce the activity of this enzyme. In chickens, endotoxins are known to be severe stressors and to elevate corticosteroids (Curtis et al., 1980). However, endotoxins also inhibit TPO activity, and this influences the metabolism of serotonin (Agarwal and Lazar, 1977; Berry and Smythe, 1963; Moon, 1985; Takikawa et al., 1986). It has been shown that this monoamine sensitizes animals to endotoxin administration (Berry and Smythe, 1963). Thus, the objective of this study was to determine the effects of B. avium infection on TPO activity in turkey poult liver homogenates and to ascertain the mechanism of action of the bacteria on TPO activity. MATERIALS AND METHODS Birds In experiment 1, 80 large white Nicholas turkey poults of mixed sexes were obtained from a commercial hatchery (Goldsboro Milling Company, Goldsboro, NC) within 6 h after removal from the hatcher. Poults were neither treated with antibiotics nor subjected to toe and beak or snood trimming. Poults were placed in metal growing batteries in environmentally controlled isolation rooms where ambient temperature was maintained at 32°C during week 1, 27°C during week 2, and 23°C during the final 2 weeks of the study, but ambient humidity varied from 45-65% over

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ANDREW G. YERSIN et al.

an experimental period of 4 weeks. Turkey 1 starter diet (North Carolina Agricultural Research Service) and water were available ad libitum. Treatments

The birds were divided randomly into two groups consisting of 40 control and 40 infected poults, and these separate groups were housed in separate rooms. The control and infected groups were subdivided into two treatment subgroups consisting of 20 saline-treated control, 20 tryptophan-treated control, 20 saline-treated infected, and 20 tryptophan-treated infected. The experimental groups were further separated into two replicates giving a total of 10 poults per subgroup. Due to mortality and culling of some poults, means were based on six poults per sub-group. Infection

One-day-old poults were infected through the delivery of 0.05 ml of a 24-hr brain heart infusion broth (Difco Lab., Detroit, MI) culture of the W isolate of B. avium (approximately 1 x 107 bacteria). The inoculum was placed on the external nares, and the poults were allowed to breathe normally inhaling the bacteria. All groUps of poults were observed daily for clinical signs of the disease and only those receiving the inoculum developed signs of coryza within 7-8 days post inoculation. Tryptophan treatment

On 3 consecutive days prior to sampling on days 7, 14, 21, and 28 post-inoculation both control and infected subgroups were given either 0.85% saline or tryptophan (Ajinomoto) (500 mg/kg) by gavage. On the sampling days, poults were bled via cardiac puncture, killed by cervical dislocation, and livers were dissected. Assays

Plasma corticosterone activity was determined by competitive protein-binding assay (Edens, 1978). TPO activity was measured in dissected livers as follows (Kim and Hill, 1967). The liver homogenate was prepared by grinding 2.0 g of liver in 10ml of 0.15 M KC1, 0.1 ml of 40mM L-tryptophan, 1.0 ml of freshly prepared 300 mM ascorbic acid and 4 vg of haemin. The solutions were adjusted to pH 7.0 before use. After removal of the debris by centrifuging at 1000g, the homogenate was recentrifuged at 100,000g for 60 min and the supernatant fluid was used as the source of the enzyme. TPO activity was determined by measuring the kynurenine formed from the oxidation and hydroxylation of tryptophan and formylkynurenine, respectively (Feigelson and Greengard, 1961). The reaction mixture contained 0.5ml of the supernatant fluid, plus 2.0ml of 0.2M Na2HPO4 buffer (pH 7.0), 0.2 ml of 40 mM L-tryptophan, 0.1 ml of 300mM ascorbate and 0.4/tg of haemin. The assays were carried out at 25°C and spectrophotometric readings at 360 #m were taken every 10 sec up to 2 min and every minute thereafter for a total of 10 min. Kynurenine concentration was calculated from the optical density of the experimental filtrate minus it's blank by the use of the molar extinction coefficient of kynurenine (E = 4530). Activity is expressed as U ~ m o l min-] g prot-l). In experiment 2, a dose study was conducted to examine the induction of TPO activity by exogenous hydrocortisone (Sigma, St. Louis, MO) administration. Birds were obtained and housed as described in experiment 1. Birds were not exposed to B. avium and were maintained until 12 days of age when they were treated with hydrocortisone via intramuscular injection. The poults (n = 24) were divided into four groups of six birds each. Each bird from the four groups received hydrocortisone at the concentration of either 0, 0.5, 1.0, or 1.5 mg in a vol of 0.25 ml dimethyl sulfoxide (DMSO) vehicle. Birds were killed 3 hr after treatment by cervical dislocation and liverswere excised and prepared for TPO assay as described in experiment 1.

Statistics

A completely randomized design was used in each experiment, and the data from these experiments were subjected to analysis of variance to distinguish time and treatment effects (SAS, 1986). Differences between treatment means were determined by Student's t-test. Statements of significance are based upon P ~<0.05. RESULTS S h o w n in Fig. 1 are the data for the response of liver T P O to exogenous hydrocortisone administration. Increasing c o n c e n t r a t i o n s of corticosterone increased the activity o f the enzyme in the liver of 12-day-old turkey poults, a n d there were significant ( P ~< 0.05) differences between the low a n d high doses administered. The activities o f liver T P O in control a n d infected poults over the 28 day experimental period are presented in Fig. 2. A t 7 days post-infection T P O activity was significantly elevated in the infected poults given t r y p t o p h a n , a n d at 14, 21 a n d 28 days post-infection, T P O activity was significantly ( P ~< 0.05) increased in b o t h control a n d infected poults, given exogenous t r y p t o p h a n , c o m p a r e d to p o u r s given the saline vehicle only. A t all sampling times over the 28 day experimental period, control a n d infected poults, not treated with t r y p t o p h a n , were f o u n d to have comparable T P O activities while the T P O activities o f b o t h control a n d infected poults given t r y p t o p h a n were elevated significantly a n d to the same magnitude. In addition to t r e a t m e n t differences for T P O activity, there was a time difference as well (Fig. 2). C o n t r o l birds treated with saline exhibited a general decrease in T P O activity over time with significant ( P ~< 0.05) differences between 7 a n d 14 days postinoculation. T P O activity in the controls c o n t i n u e d to decrease at 21 a n d 28 days. C o n t r o l poults treated with t r y p t o p h a n also showed a numerical decrease in T P O activity over time, but differences were n o t significant until 28 days post-inoculation. The responses o f infected poults treated with saline were similar to infected poults treated with t r y p t o p h a n . T P O activity was significantly ( P ~< 0.05) reduced at days 14 a n d 21 p o s t - i n o c u l a t i o n c o m p a r e d to days 7 a n d 28 post-inoculation in b o t h groups. Tryptophan

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Fig. 1. Tryptophan 2,3-dioxygenase (TPO) activity (#mol min -~ g prot -t _+ SEM) in liver homogenates following exogenous hydrocortisone administration in DMSO vehicle. Different lower case letters in the bars on the graph indicate significant differences (P ~<0.05).

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Fig. 2. Tryptophan 2,3-dioxygenase (TPO) activity ~mol/min-t g prot-I + SEM) in liver homogenates from control and infected poults treated with tryptophan or saline. Different lower case letters in the bars on the graph indicate differences (P ~<0.05).

The plasma corticosterone concentration of all poults from both treatment groups during the four sampling times are illustrated in Fig. 3. There were no significant differences in corticosterone concentrations between control and infected poults or among treatment groups within any sampling time. However, in control poults, corticosterone concentration decreased significantly across the entire experimental period, but the infected poults showed a peak in corticosterone at day 14 post-infection followed by a decreasing concentration on days 21 and 28. Over all times of sampling, the infected poults maintained a significantly higher mean corticosterone than d i d the controls. Tryptophan did not elevate the plasma corticosterone concentration in either control or infected birds. DISCUSSION

Tryptophan 2,3-dioxygenase is an iron porphyrin enzyme which catalyzes the oxidative cleavage of

Plasma

L-tryptophan to N-formylkynurenine and exists in three forms: an apoenzyme, as well as an oxidized and reduced holoenzyme (Moon, 1985). Both the apoenzyme and the oxidized holoenzyme require further activation to become catalytically active as the reduced form and the overall activation process requires the presence of the substrate-L-tryptophan, the prosthetic group-hematin, and a reducing agent such as ascorbic acid or hydrogen peroxide (Badawy and Evans, 1975; Greengard and Feigelson, 1961; Knox and Piras, 1966; Moon, 1985). The enzyme is synthesized in the liver and is important for the gluconeogenic response as indicated by increased activity after glucocorticoid treatment (Moon, 1985; Peterkofsky, 1968; Renkawitz et HI., 1984). It appears that both the substrate (tryptophan) and the glucocorticoids induce enzyme activity but by different mechanisms. Administration of L-tryptophan induces activity resulting from a decreased rate of degradation of the enzyme, while glucocorticoids induce the enzyme by a mechanism which involves new R N A

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ANDREWG. YERSINet al.

formation and an increased rate of formation of enzyme (Badawy and Evans, 1975; Moon, 1985; Peterkofsky, 1968). This study demonstrated that TPO activity in turkey poults was influenced by substrate (tryptophan) loading. Tryptophan treatment for 3 consecutive days caused an increase in enzyme activity in both control and infected poults. This may have stabilized the degradation of enzyme present and increased the amount of apoenzyme being formed. In the present experiment, the dosage (500 mg/kg) was given because tryptophan is rapidly metabolized and excreted making substrate induction of TPO shorter lived than hormonal induction. In studies with mice, treated with endotoxin, as much as 240 rag/mouse was given over a 24 hr period to maintain TPO levels (Moon, 1985). B. avium infection has been characterized as a stressor (McCorkle et al., 1985; Saif et al., 1980; Simmons et al., 1979), and as a stressor it should have induced TPO. Observations in this study included reduced weight gain, watery diarrhoea, reduced water and food intake, and a general lethargy in infected poults. It was reported that B. avium infection significantly elevated serum corticosterone (McCorkle et al., 1985). These physiological responses to B. avium infection should have increased the activity of the enzyme, but this effect was not observed in the study. Therefore it is possible to speculate that the lack of an increase in the TPO activity in the infected, saline-treated poult was attributed to a lack of available substrate, tryptophan, and not due to a lack of the corticosteroid inducer. Even though there were no significant differences in corticosterone concentrations among treatment groups within any sampling period, and one can speculate that the repeated handling of all poults for 3 consecutive days, and the gavage treatments caused an elevation in corticosterone in all treatment groups. This would be consistent with an acute stress response in these animals. Although no significant differences within a sampling period were found, it is important to note that control poults reduced plasma corticosterone concentrations over the 28 day period, and the infected poults showed a peak in concentration at day 14 post-infection when clinical signs are usually at their greatest and a numerically sustained elevation in corticosterone over the entire experimental period. This observation indicated that the adrenal cortex was secreting corticosterone maximally in the infected poults, and further stressing by gavage treatments did not increase the adrenal secretory rate. It is also possible that the failure to achieve a significant rise in corticosterone in the infected birds above the control birds was due to an inability of the endogenously produced corticosteroids to be further elevated with the application of an additional stressor. A similar effect in heat stressed ACTH-treated chickens has been reported (Edens, 1977, 1978). Thus, these observations lead to the conclusion that there is a lack of substrate available in the infected poults which prevents the normal rise in TPO activity. Alternatively, endotoxins have been reported to inhibit the activity of tryptophan 2,3-dioxygenase in mice (Moon, 1985). Because tryptophan 2,3-dioxygenase is the first enzyme in the pathway leading to

the formation of pyridine nucleotides, its depression implies a block in the biosynthesis of these compounds, and has biological significance since both N A D and nicotinamide protect mice against endotoxin lethality (Moon, 1985). The role endotoxin plays in B. avium infection has not been determined, but the existence of the role has been confirmed (Edens et al., 1984; McCorkle et al., 1985; Simmons et al., 1986). The lack of the expected increased activity of tryptophan 2,3-dioxygenase in the infected poults suggested that endotoxin may have been involved. This suggestion is supported by the fact that enzyme activity was lowest in the infected-salinetreated birds at days 14 and 21 post-infection which corresponds to the time period in which clinical infection is generally most intense (Simmons, 1984). The slight depression in enzyme activity in infected poults, although not significantly different from controis, may be indicative of an increase in a biochemical pathway leading to other tryptophan metabolic products such as serotonin synthesis. Serotonin is synthesized in the central nervous system and intestine from tryptophan and is distributed throughout the body, primarily by platelets in mammals and by thrombocytes and eosinophils in birds (Emerson, 1985; Sturkie, 1986). It is known that serotonin sensitizes animals to endotoxin (Berry and Smythe, 1963). It is also known that B. avium infection depresses whole brain serotonin concentrations (Edens et al., 1987), but no studies have examined serum or plasma serotonin concentrations during infection. It is possible that the reduction in TPO activity, during infection, enhances serotonin release from thrombocytes and eosinophils causing greater sensitization to the endotoxin and the bacterial infection itself. The data also suggest that endotoxin effects may have been blocked partially or masked by induction of the enzyme via substrate presentation since infected poults, treated with tryptophan, showed an elevation in enzyme activity similar to or greater than control tryptophan-treated poults. This response may be the result of increased synthesis of N A D via excess tryptophan availability. While the effects of B. avium infection cannot be attributed entirely to endotoxin, the possibility that it may play a role in the altered physiological state of the poult cannot be dismissed. The reduced activity of the enzyme may be contributing to other physiological mechanisms which make the poult more susceptible to secondary pathogens and to the general stress which is apparent in the infected poult.

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Tryptophan 2,3-dioxygenase activity in turkeys Civen M. and Knox W. E. (1959) The independence of hydrocortisone and tryptophan inductions of tryptophan pyrrolase. J. biol. Chem. 234, 1787-1790. Curtis M. J., Flack I. H. and Harvey S. (1980) The effect of Escherichia coli endotoxins on the concentrations of corticosterone and growth hormone in the plasma of the domestic fowl. Res. Vet. Sci. 28, 123-127. Edens F. W. (1977) Physiological profile of heat prostration in chickens. Proc. South Reg. Avian Environ. Physiol. Bioeng. Study Group 13, 34 53. Edens F. W. (1978) Adrenal cortical insufficiency in young chickens exposed to a high ambient temperature. Poultry Sci. 57, 1746-1750. Edens F. W., McCorkle F. M. and Simmons D. G. (1984) Body temperature response of turkey poults infected with Alcaligenes faecalis. Avian Pathol. 13, 787-795. Edens F. W., McCorkle F. M., Simmons D. G. and Yersin A. G. (1987) Brain monoamine concentrations in turkey poults infected with Bordetella avium. Avian Dis. 31, 504-508. Emerson T. E. (1985) Release and vascular effects of histamine, serotonin, angiotensin II, and renin following endotoxin. In Handbook of Endotoxin, Vol. 2, Pathophysiology of Endotoxin (Edited by Hinshaw L. B.), pp. 173-202. Elsevier, Amsterdam. Feigelson P. and Greengard O. (1961) A microsomal ironporphyrin activator of rat liver tryptophan pyrrolase. J. biol. Chem. 236, 153-157. Fernstrom J. D. and Wurtman R. J. (1971) Brain serotonin content: physiological dependence on plasma tryptophan levels. Science 173, 149-151. Gentry-Weeks C. R., Cookson B. T., Goldman W. E., Rimler R. B., Porter S. P. and Curtis R. III (1988) Dermonecrotic toxin and tracheal cytotoxin, putative virulence factors of Bordetella avium. Inf. Imm. 56, 1698-1707. Greengard O. and Feigelson P. (1961) The activation and induction of rat liver tryptophan pyrrolase in vivo by its substrate. J. biol. Chem. 236, 158 161. Kim C. S. and Hill C. H. (1967) The effect of copper deficiency on the induction of tryptophan pyrrolase in chick liver by hydrocortisone. Biochem. J. 103, 256-257. Knox W. E. and Auerbach V. H. (1955) The hormonal control of tryptophan peroxide in the rat. J. biol. Chem. 214, 307-313. Knox W. G. and Piras M. M. (1966) A reinterpretation of the stabilization of tryptophan pyrrolase by its substrate. J. biol. Chem. 241, 764-767.

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