J APPL POULT RES 2006. 15:183-189
© 2006 Poultry Science Association
Dietary Lutein Influences Immune Response in Laying Hens
G. Y. Bédécarrats1 and
S. Leeson
Department of Animal and Poultry Science, University of Guelph, Guelph, Ontario, Canada, N1G 2W1
Correspondence: 1 Corresponding author: gbedecar{at}uoguelph.ca
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SUMMARY
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Lutein, a xanthophyll carotenoid, has been proposed as a potential candidate for the prevention of numerous diseases in human. Because lutein content in eggs is directly correlated with the lutein content in hen diets, there is interest in producing a lutein-enriched designer egg. Evidence in several species also suggests direct effects of lutein on the immune response. Because flaxseed is commonly used in layer diets, we investigated the potential effects of dietary lutein supplementation with or without flaxseed supplementation on phytohemagglutinin P (PHA-P)-induced cutaneous hypersensitivity and on antibody response to infectious bronchitis vaccination in layers. No significant effect was observed on cutaneous hypersensitivity to PHA-P, but dietary lutein significantly boosted the secondary antibody response to infectious bronchitis vaccination. Flaxseed supplementation alone did not significantly affect the parameters studied. However, reduced lutein effect was observed when lutein was combined with flaxseed. In conclusion, dietary lutein stimulates the antibody response to infectious bronchitis virus vaccination. Thus, in addition to producing designer eggs, lutein supplementation also likely benefits the health of the flock by increasing the efficacy of vaccination.
Key Words: chicken • layer • lutein • immune system • infectious bronchitis
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DESCRIPTION OF PROBLEM
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Lutein is a carotenoid naturally found in marigold, corn, and dark-green vegetables. Recent evidence suggests lutein could be beneficial in the prevention of a wide array of human diseases (for review, see [1]). However, studies in Europe and North America have determined the average daily intake of lutein in adult humans to be insufficient; to provide a readily available source of lutein, efforts have recently focused in enriching eggs by supplementing laying hen diets [2]. Efficient transfer of lutein into eggs is affected by dietary composition [2], and large-scale studies are currently underway in our facility to determine optimum conditions. Although the beneficial effects of lutein have mainly been studied in humans, recent evidence also suggests it affects the immune response in other animals, such as cats [3] and dogs [4]. In the present study, we investigated the effect of dietary lutein supplementation on cutaneous hypersensitivity and antibody response in layers. Because most current designer eggs are produced by birds fed flaxseed, we also investigated the potential for interactions of flaxseed and lutein on these immune parameters.
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MATERIALS AND METHODS
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Birds and Diets
All of the experiments described in this study were covered by an animal use protocol obtained from the University of Guelph animal care committee.
One-day-old White Leghorns [5] were obtained from a commercial hatchery. Birds were raised under an 8-h photoperiod until 18 wk of age, and then pullets (n = 300) were transferred to individual battery cages, raised under a 14-h photoperiod, and randomly separated into 6 experimental groups (n = 50 per group). The birds were individually fed ad libitum with a standard diet formulated to meet or exceed NRC [6] specifications. At 19 wk of age, a layer diet was supplemented with 125 or 250 ppm lutein (provided as OroGlo, 15 g of active product/kg [7]), flaxseed, or both as described in Table 1
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Immunity response tests were performed at 39 wk of age on hens that had been fed the experimental diets for 20 wk. At that time, mean egg production exceeded 90%. For each experiment (cutaneous hypersensitivity and antibody response), 10 hens were randomly selected from within each of the 6 experimental treatments.
Cutaneous Hypersensitivity
Phytohemagglutinin P (PHA-P) has previously been reported to stimulate cutaneous basophil hypersensitivity and lymphoproliferation in broiler and layer chickens [8, 9]. The procedure followed was based on the methodology described by Corrier [9]. Briefly, hens were injected intradermally between the third and fourth digits of the left foot with 100 µg of PHA-P in 0.1 mL of saline solution. As a control, 0.1 mL of saline solution was injected into the right foot following the same procedure. Cutaneous hypersensitivity was evaluated by measuring toe web thickness before injection and at 24 and 48 h after; measurements were taken with a micrometer with an accuracy of 0.01 mm.
Antibody Response
All birds were subjected to a standard vaccination protocol. One-day-old chicks were sprayed with live infectious bronchitis virus (IBV) vaccine [5] and injected with Marek’s disease live virus vaccine [5]. At 4 and 11 wk of age, birds were vaccinated with an eye drop version of the Newcastle bronchitis live virus vaccine [5]. Finally, at 16 wk of age, pullets were injected intramuscularly with a Newcastle bronchitis killed virus vaccine [5].
To determine if dietary lutein influenced antibody response to secondary IBV exposure, birds from each of the 6 experimental treatments (n = 10 / diet) were dosed by eye drop with live IBV vaccine (Merial Select). Blood samples were collected 1 d before vaccination and at 7, 14, and 28 d after. Plasma was separated by centrifugation and frozen at –80°C until subsequent assay for antibody titer. Antibody testing was done [10] using the FlockChek Infectious Bronchitis Virus Antibody Test Kit [11]. A hen was considered as responding to vaccination when the antibody titer was increased by at least 10% at any time point after exposure. A hen was considered as not responding when the antibody titer did not increase by at least 10% after vaccination.
Statistical Analysis
All statistical analyses were performed using INSTAT 3.0 [12]. Analyses of cutaneous hypersensitivity and antibody response within each experimental group (between pre- and postinjections) were performed using a repeated measures ANOVA followed by a Tukey-Kramer post hoc test. Comparisons among experimental treatments for individual time points were performed by ANOVA followed by a Tukey-Kramer post hoc test. Significance was assigned at 95% confidence (P < 0.05).
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RESULTS AND DISCUSSION
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In humans, several studies have suggested that lutein may help prevent the occurrence of a number of diseases, ranging from macular degeneration to cancer [1]. A direct effect of lutein on the immune system has also been reported in mice [13, 14]. Similarly in dogs, dietary lutein has been shown to stimulate PHA-induced lymphoproliferation and to stimulate antibody response to a secondary antigen exposure [4]. As reviewed by Chou and Park [15], the effect of lutein on the immune system might reside in its antioxidative properties. Koutzos et al. [16, 17] reported that lutein is present in immune cells of chickens and that carotenoid deposition in immune tissues of growing chicks is influenced by maternal diet composition, indicating that dietary lutein could be efficiently transferred from breeder hens to their offspring. Furthermore, direct in ovo injection of lutein in nestling barn swallows enhances T-cell-mediated immune response [18]. However, the facilitating effect of dietary lutein on adult laying hen immune responses remains to be determined.
Cutaneous Hypersensitivity to PHA-P
Injection of saline solution alone did not induce any significant increase in skin thickness (data not shown). As previously reported for young layers and broilers [8, 9], 100 µg of PHA-P injected significantly (P < 0.01) increased toe web skin thickness within 24 h after injection in all our experimental groups, and except in birds fed flaxseed with 250 ppm lutein swelling remained significant (P < 0.05) after 48 h vs. prior to injection (Figure 1). Conversely to what was reported in mice [13], we did not observe any significant effect of lutein on cutaneous hypersensitivity. In a previous study in dogs, lutein supplementation for 6 wk resulted in a significant dose effect on PHA challenge 24 h after injection [4]. However, when lutein was given for 12 wk, only the highest dose produced a significant response at 48 and 72 h postinjection [4]. In our study, hens were supplemented with lutein for 20 wk before PHA-P injection, and it is possible a stimulatory effect on cutaneous hypersensitivity could no longer be observed.
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Figure 1. Effect of dietary lutein supplementation on phytohemagglutinin P (PHA-P)-induced cutaneous hypersensitivity. Hens (n = 10 per group) fed experimental diets for 20 wk were injected intradermally between the third and fourth digits with 100 µg of PHA-P in 0.1 mL of saline solution. Toe web thickness was measured before (black bars), 24 h after (gray bars), and 48 h after (white bars) injection. Results are expressed as mean thickness ± SEM *,**Significant differences with the preinjection value at P < 0.05 and P < 0.01, respectively.
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Antibody Response
Secondary antibody response to a live IBV vaccine is shown in Table 2. Even if most experimental groups displayed increased antibody titers, a significantly (P < 0.001) rapid and sustained response was observed only in birds fed 125 ppm lutein, although no such response was observed with 250 ppm lutein. Antibody titer also increased significantly (P < 0.05) when 10% flaxseed was combined to 125 ppm lutein in the diet. However, this increase was not as rapid nor as sustained as observed for birds fed 125 ppm lutein only. One of the most striking results was the number of birds that responded to the vaccine within each experimental group. Of the birds fed flaxseed or 250 ppm lutein diets, 50% failed to respond to the vaccine, and only 60% of the birds fed the control diet or flaxseed plus 250 ppm lutein had increased antibody titers. Conversely, 80% of the hens fed 125 ppm lutein with flaxseed and 90% of the hens fed 125 ppm lutein without flaxseed responded to immunization. Interestingly, most of the hens that did not have an increase in antibody titers when on the diet with flaxseed or 250 ppm lutein had high antibody titers before vaccination. It is thus possible that these high initial levels prevented our live vaccination from triggering a response. However, initial titers from hens that did not respond in the other experimental groups did not differ from the responding birds. To account for these differences, mean antibody titer values from responsive birds were calculated and are shown in Table 3. Even if no difference in antibody titers was observed among diets, a rapid and sustained significant response was observed in both groups fed 125 ppm lutein (with or without flaxseed). A significant response for shorter duration (d 7 and 14) was also observed in chickens fed 250 ppm lutein. The effect on antibody titers at d 7 and 14 in the responsive birds fed the control diet was not significant (P > 0.05). The combination of flaxseed with 250 ppm lutein resulted in the poorest response. Although the base levels of antibody titers were not significantly different prior to vaccination, large variations in values were observed between individuals. To accurately compare the effect of any diet on antibody response, titers for each responsive bird were normalized to their initial value (pre-immunization), and results were expressed as fold increase (Figure 2). Hens fed 125 ppm lutein had antibody titers significantly higher than hens fed the control diet at d 7 and 28, and titers were significantly higher than for hens fed the flaxseed with 250 ppm lutein diet at d 14 and 28. The increase in antibody titers for hens fed the diet with 125 ppm lutein and flaxseed also seemed to follow the same trend as the group fed 125 ppm lutein. No significant differences, however, were observed in this instance.
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Table 2. Effect of dietary lutein on infectious bronchitis virus (IBV) antibody titers in hens challenged with a live vaccine1
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Table 3. Effect of dietary lutein on infectious bronchitis virus (IBV) antibody titers in responsive hens challenged with a live vaccine1
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Figure 2. Dietary lutein stimulates infectious bronchitis virus (IBV)-specific antibody production in responsive hens challenged with a secondary vaccination. Hens fed experimental diets for 20 wk were exposed to a secondary IBV vaccination. Antibody titers were measured in plasma before vaccination (d 0) and at 7, 14, and 28 d after vaccination, and titers from responsive hens (control, n = 6; lutein 1, n = 9; lutein 2, n = 5; flax, n = 5; flax/lutein 1, n = 8; flax/lutein 2, n = 6) were corrected to their initial value. Results are expressed as mean fold titer increase ± SEM. *Significantly different mean values (P < 0.05).
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In mice, lutein has previously been shown to enhance the response of B lymphocytes to stimulation in vitro and in vivo [14, 15], and in dogs and cats [3, 4] lutein increases circulating antibody concentrations. In our study, the amplitude of the secondary antibody response to a live IBV vaccine was significantly enhanced in hens fed lutein, especially at 125 ppm. At this dosage, high levels of IBV antibodies were sustained for at least 28 d (last time point examined). We did not evaluate the effect of the diet on primary response. However, no significant difference in antibody titer was observed the day before immunization. Under commercial conditions, it is well accepted that layer flocks generally have a relatively poor response to IBV vaccination, and it is not uncommon to observe only 50% of the birds responding. Interestingly, in our study, the number of animals that did respond to the vaccine by displaying an increase in antibody titer was higher in the birds fed 125 ppm lutein (with or without flaxseed). This observation suggests that lutein increases the sensitivity of the humoral immune system, maybe by enhancing T-helper type-2 cells, intimating a larger pool of memory cells, or as suggested for dogs [4], by enhancing cytokine response and antigen-presenting cells activity. Alternatively, it is also possible that the replication of the live virus used for vaccination was affected by the diets, thus altering the amplitude of the immune response.
Because a dose-dependent effect of lutein has previously been reported in other species, we were surprised to observe that the high dose of lutein (250 ppm) failed to significantly boost the antibody response to IBV vaccination. In human dendritic cells, excess antioxidant can down-regulate the immune response [19], and because lutein likely acts as an antioxidant, it is possible that at high doses it prevented the initial increase in reactive oxidant species necessary to stimulate lymphocytes. Alternatively, it is also possible that high lutein content in the diet affects its assimilation. In previous studies [2, 20] Leeson has shown feed-to-egg transfer efficiencies of 10 and 5% with lutein supplements of 125 and 250 ppm, respectively, which are the levels used in the current study.
Flaxseed is now commonly added to commercial layers diet, and although we did not observe any significant effect of flaxseed alone on IBV antibody titer, combining lutein with flaxseed seemed to reduce the positive effect of lutein on the IBV antibody concentration as well as the PHA-P-induced cutaneous hypersensitivity. Enrichment of broilers diet with flaxseed has previously been shown to decrease antibody-dependent cell cytotoxicity by splenocytes but not by peripheral blood leukocytes [21]. However, in the same study, flaxseed supplementation did not alter the primary or secondary antibody response to sheep red blood cells. It has also been reported that diet composition can alter layers’ splenocyte content in n-6 and n-3 polyunsaturated fatty acids and that decreasing the n-6 to n-3 ratio reduces lymphoproliferation, whereas it could increase IgG concentration in serum [22]. However, the source and type of n-3 polyunsaturated fatty acid also appear to be important, as fish oil and linseed oil are more potent in reducing lymphoproliferation and promoting higher IgG levels, respectively [22]. In our study, reduction of the n-6 to n-3 ratio did not stimulate the antibody response to IBV vaccination but rather tended to attenuate the positive effect of lutein. In layers, a significant reduction in lutein transfer from the diet to the egg was also observed with the addition of flaxseed, especially at higher levels of lutein supplementation [2]. It is thus possible that in addition to a potential negative interaction, flaxseed interfered with lutein digestibility.
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CONCLUSIONS AND APPLICATIONS
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- This study reports a beneficial effect of dietary lutein on the immune system of adult laying chickens in which lutein significantly boosted the secondary antibody response to live IBV vaccination.
- In addition to its role as a neutraceutical in designer eggs, lutein could also likely benefit the immune response of laying hens.
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ACKNOWLEDGMENTS
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This research was supported by the Canadian Egg Marketing Agency (Ottawa, ON), the Ontario Egg Producers (Mississauga, ON), the National Science and Engineering Research Council (Ottawa, ON), Kemin Industries (Des Moines, IA), and the Ontario Ministry of Agriculture and Food (Guelph, ON).
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REFERENCES AND NOTES
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SUMMARY
DESCRIPTION OF PROBLEM
