J.C. LOPEZ
R. MCFARLANE
O. AMOAFO
Agriculture and Life Sciences Division,
Lincoln University, Lincoln, Canterbury,
New Zealand
The effect of environmental stressors on the immune system of broilers was mimicked by the administration of corticosterone (CORT). Commercial broilers were randomly assigned to one of four treatment groups (n=50):
- group 1 [not challenged with infectious bronchitis virus (IBV) or injected with exogenous antigen but treated with corticosterone (CORT) from 23 days of age];
- group 2 [challenged with IBV, injected with sheep red blood cells (SRBC) and phytohaemagglutinin (PHA) but not treated with CORT];
- group 3 (challenged with IBV, injected with SRBC and PHA and treated with CORT from 23 days of age), and
- group 4 (challenged with IBV, injected with SRBC and PHA and treated with CORT from 26 days of age).
The birds given CORT in the drinking water from five days before injection with SRBC (23 days of age), showed an enhancement in the humoral immune response, in comparison to the birds given CORT three days later (two days before challenge) or the birds that were untreated with CORT. Furthermore, the birds given CORT five days before IBV challenge, developed a higher cell mediated immune response as measured by the level of INFγ released by splenic lymphocytes, after in vitro stimulation with IBV. That is, the immunological effects of administering physiological amounts of CORT, which may reflect temperature extremes, are dependent on timing relative to exposure with environmental antigen.
Introduction
Extremes of ambient temperature are important stressors that confront poultry in many regions of the world and large economic losses can occur because of mortality and decreased production (Altan et al., 2000). Results from studies on the effect of temperature extremes on humoral and cell-mediated immune responses in chickens are inconsistent (Heller et al., 1979, Subba Rao and Glick, 1970). We have recently shown that low temperatures (10°C) can cause elevated blood levels of CORT (unpublished data). It has been proposed that the time between the release of CORT and challenge is a factor that can change the immune response (towards inhibition or enhancement) (Dhabhar, 2002). The goal of this study was to assess the effect of administering physiological doses of CORT on humoral and cell-mediated immune responses in chickens, following infection with IBV and injection with SRBC and PHA.
Materials and methods
Two hundred, day-old broiler chickens (Cobb breed) were obtained from a commercial hatchery. At 23 days of age the birds were randomly assigned to one of four treatment groups (n=50):
- groups 1 and 3 [treated with CORT (14.7 ng/ml) in the drinking water from 23 days of age (five days before challenge)];
- group 2 (not treated with CORT); and
- group 4 [treated with CORT in the drinking water (14.7 ng/ml) from 26 days of age (two days before challenge)].
At 28 days of age all birds in groups 2, 3 and 4 were infected intranasally with 100μl of 106 EID50/ml of IBV (NZ strain C - supplied by the National Centre for Disease Investigation, Upper Hutt, New Zealand), and injected with 0.5 ml of packed sheep red blood cells (50% v/v in PBS) into the breast muscle. Additionally, at 45 days of age, eight birds from groups 2, 3 and 4 were injected in the right wattle with 200 µg of PHA in 0.1ml of sterile, pyrogen-free, physiologic saline solution, while the left wattle was injected with saline as a control. The wattle thickness of each bird was measured with a dial micrometer 24h later, as described by Tella et al. (2002).
Antibody production against SRBC was measured using a micro haemagglutination test (1:2 dilution), as described by Wegmann and Smithies (1966) and results were expressed as the reciprocal of the highest dilution of serum showing specific agglutination with antigen.
Twelve birds from each of the four groups were slaughtered at 23, 28, 38, and 43 days of age in order to assess the levels of INFγ from splenocytes that had been separated on Ficoll-PaqueTM Plus (Amersham Biosciences, Sweden), washed twice in RPMI 1640 medium (Sigma-Aldrich, USA), and resuspended in the same medium supplemented with 10% FCS.
Cells, at a concentration of 1x 107/ml, were incubated at 39oC for 72 hours with IBV antigen whole virus at 10 μg/ml, as per Lambrecht et al. (2000). Supernatants were collected after centrifugation at 3,000 rpm for 15 minutes and stored at -70oC. The levels of INFγ in the supernatant were estimated using a sandwich ELISA (Biosource International, USA).
The CORT levels were measured by radioimmunoassay (RIA), at the Institute of Veterinary, Animal and Biomedical Sciences, Massey University (Littin and Cockrem, 2001). Significance was assessed using an analysis of variance, and differences between the means were determined using the Least Significant Difference (Minitab Statistical Software).
Results and discussion
By 28 days of age, the birds, from Group 1 and 3, that started the CORT treatment five days before challenge (23 days of age) had a significantly higher (P<0.01) level of plasma CORT than the birds not treated with CORT (group 2). At 36 days of age the birds from the groups treated with CORT (groups 1, 3 and 4) had significantly (P<0.01) higher levels of plasma CORT than the birds not treated with CORT (group 2) (Figure 1a). These CORT levels are similar to levels found in poultry housed at low (10°C) temperatures (unpublished data).
Eight days after injection with SRBC it was found that the chickens treated with CORT from 23 days of age (five days before challenge) had a significantly higher HA titre (P<0.01) than the birds not treated with CORT, which in turn developed a higher HA titre (P<0.05) than the birds treated with CORT from two days before challenge (26 days of age) (Figure 1b). Moreover, the birds that were given CORT in the drinking water five days before the challenge developed a higher cell mediated immune response, as measured by the level of INF-γ released by splenic lymphocytes after in vitro stimulation with IBV, in comparison to the birds exposed to CORT for two days before challenge (Figure 1c).
In humans, exposure to cortisol for up to a week before a challenge with endotoxin enhances TNF- α and IL-6 levels, whereas cortisol at the time of or after endotoxin suppresses the cytokine response (Barber et al., 1993). The skin reaction to PHA injected 24 hours previously (49 days of age) showed that the birds from the groups treated with CORT, either two or five days pre¬challenge, had significantly smaller reactions than untreated birds. This in vivo test provides a general index of cell mediated immunity (Tella et al., 2002), as compared to the in vitro, antigen-specific (IBV) assay measuring INFγ production from T cells present in the spleen (Lambrecht et al., 2000).
It has been argued that the immunological actions of CORT are vested in the basal levels of CORT in blood at the moment of challenge and possibly the pre-exposure period, causing down-regulation of glucocorticoid receptors or alteration of their affinity (Armario et al., 1994). In this experiment the transient immuno-enhancement was associated with the longer duration of steroid intake (five days) and immuno-suppression occurred with the shorter duration (two days). Plasma CORT levels did not differ at the time of challenge due to timing of administration. In an experiment described by Post et al. (2003) the immunosuppressive effects of the exogenous CORT were noted after three days of CORT treatment achieving very high plasma CORT concentrations (30-35 ng/ml) in comparison to the physiological levels reached in this experiment (9.5 ng/ml). Enhancement of the immune response may occur via an increase in cytokine receptor expression on several cell types (e.g., T cells) for IFNγ (Strickland et al., 1986), or alternatively, by leukocyte redistribution (Dhabhar, 2002).
The majority of the studies on corticosteroids have focused on the immunosuppressive actions of these stress hormones. In this experiment it was found that in chickens, as for other species, the time between the beginning of the CORT treatment and the moment of challenge is crucial in determining response. This is consistent with the view that corticosteroids can exert distinct, seemingly paradoxical, effects on cytokine expression, cytokine receptor expression and cytokine-regulated biological responses (Wiegers and Reul, 1998).
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From Proceedings of the "19th Australian Poultry Science Symposium", New South Wales, Australia.
