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IMMUNOLOGY, HEALTH, AND DISEASE |
Adaptation Physiology Group, Department of Animal Sciences, Wageningen Institute of Animal Sciences, Wageningen University, 6700 AH Wageningen, The Netherlands
1 Corresponding author: Henk.Parmentier{at}wur.nl
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: chicken • immune response • pathogen-associated molecular pattern • lipopolysaccharide • 1,3 ß-glucan
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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1,3 ß-Glucans are shown to enhance inflammatory cytokine production and activation of complement without immunoglobulins (Frasnelli et al., 2005). Exposure to molds could deteriorate an existing inflammation in the lungs induced by inflammatory agents such as smoke, air pollution, or microbial infection (Sjöstrand and Rylander, 1997). In the presence of a strong stimulus, BGL can down-regulate harmful immune hyperactivity (Pelizon et al., 2003). On the other hand, i.t. administration of BGL (from bakers’ yeast) induces pulmonary inflammation in rats (Young et al., 2001).
Previously, we described immune modulating activities of LPS and LTA when administered intravenously in layers shortly before primary and secondary s.c. immunization with model antigens (Parmentier et al., 2004; Maldonado et al., 2005). In short, LTA enhances, whereas LPS depresses specific primary antibody responses to model T-cell-dependent antigens such as keyhole limpet hemocyanin, BSA, and rabbit 
globulin. This suggested skewing of the immune response of chickens to either T-helper-1 (TH-1) or T-helper-2-(TH-2) like immune reactivity, via binding of the PAMP to toll-like receptors (TLR) present on dendritic cells, as has been described in mammals (Dabbagh et al., 2002; Matsui and Nishikawa, 2002; Kapsenberg 2003). This may result in the release of proinflammatory TH-1 cytokines [interleukin-12 (IL-12)] or, alternatively, differentiation of the immune response toward the TH-2 route (Bliss et al., 1996; Werling and Jungi, 2002; Schroder et al., 2003). In mice, LPS is found to react via TLR-4 (Calkins et al., 2002; Werling and Jungi, 2002; Rocksen et al., 2004; Talreja et al., 2004). However, LPS was also reported to act via TLR-2, in a CD-14 dependent fashion (Schwandner et al., 1999). Lipoteichoic acid may activate cells via TLR-2 (Werling and Jungi, 2002; Lee et al., 2004; Talreja et al., 2004), and BGL is found to react via TLR-2 and probably other receptors (Frasnelli et al., 2005).
Chickens either inhale (approximately 1 m3 of air/24 h) PAMP or may obtain them via the cloaca. Concentrations of airborne endotoxins (LPS) and BGL ranging from 240 to 13,400 endotoxin units/m3 (1 endotoxin unit/m3 
0.1 ng/m3) were found in chicken farms (Anonymous, 1998, Douwes, 1998). Previously, we found also that i.t. challenge of 12-wk-old layer chickens affected specific primary and secondary antibody responses to s.c. administered antigen, suggesting the possibility of systemic immune modulation by airborne PAMP.
Five questions were pertinent for the present study: 1) which PAMP at what age modulated humoral immune responses and BW gain, 2) is modulation by PAMP prolonged in time, 3) do birds adapt to repeated exposure with the same PAMP, 4) is modulation (ir)reversible of nature, and 5) do breeds differ in responsiveness to PAMP? The same birds were exposed to 1 of 3 types of PAMP exposure at 7 wk of age, next to a control (PBS) exposure and were exposed to the same or another (crossover) PAMP at 13 wk of age. Pathogen-associated molecular patterns were administered during 5 consecutive days before primary and secondary s.c. immunization with human serum albumin (HuSA) at 7 and 13 wk of age and primary immunization with rabbit -globulin (RGG) at 13 wk of age. In earlier studies, we found prolonged effects of PAMP treatment on subsequent immune responsiveness. Therefore, we studied whether immune modulation at a young age resulted in an (ir)reversible type of immune responsiveness at a later age. In addition, effects of airborne PAMP on BW (gain) as a parameter of an acute phase response, and feed conversion (FC) were measured at both ages.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Reagents
Escherichia coli-derived LPS (L2880, serotype 055:B5), BGL derived from Saccharomyces cerevisiae, (Zymosan A, Z-4250), RGG (lot 40H9301), HuSA (lot 8763) were from Sigma-Aldrich Inc. (St. Louis, MO).
Experimental Design
At 7 wk of age, chicks received intratracheally (primary challenge) 0.5 mL of PBS (pH 7.2) containing either 0.5 mg of LPS; 0.5 mg of BGL; 0.25 mg of LPS + 0.25 mg of BGL, respectively; or only 0.5 mL of PBS (control group). Challenges were performed by placing a 1.2 x 60 mm blunted anal canule (InstruVet, Cuijk, The Netherlands) put on a 1-mL syringe gently in the trachea of the chick. The birds received the same exposure treatment during 5 consecutive days. At 13 wk of age, birds were exposed to the same PAMP or were treated with the other PAMP or PBS. At the fifth treatment day, both after 7 and 13 wk of age, all birds were immunized s.c. with 1 mg of HuSA dissolved in 1 mL of PBS. At 5 d before the first day of i.t. challenge (i.e., 9 d before), and at 3, 7, 14, 21, and 28 d after primary immunization with HuSA, blood was taken from the chickens. Similarly, blood was taken at the first day of the secondary i.t. challenge and at 3, 7, 14, 21, and 28 d after secondary immunization with HuSA. Also at the fifth day of the second i.t. exposure with PAMP, all birds were also immunized s.c. with 1 mg of RGG to mount a primary immune response at that age. Plasma was stored at –20°C until use. The BW of the chickens was recorded at various time points, from which the growth was calculated. Feed conversion during the first weeks of primary or secondary exposure to PAMP and the third week after exposure was derived from growth during these periods of 7 d divided by feed uptake. The experiment was approved by the Animal Care Committee of Wageningen University.
Humoral Immune Response to HuSA and RGG
Total specific antibody titers to HuSA and natural antibody titers to RGG in plasma from all birds were determined by ELISA at d –9, 3, 7, 14, 21, and 28 after primary immunization at 7 wk of age and at d 0, 3, 7, 14, 21, and 28 after secondary immunization with HuSA at 13 wk of age. Total specific primary antibody titers to RGG in plasma from all birds were determined by ELISA at d 0, 3, 7, 14, 21, and 28 after primary immunization with RGG at 13 wk of age. Briefly, 96 well plates were coated with either 4 µg/mL of HuSA or 4 µg/mL of RGG, respectively. After subsequent washing with H2O containing 0.05% Tween, the plates were incubated with serial dilutions of plasma. Binding of total chicken antibodies to HuSA or RGG was detected using 1:20,000 diluted rabbit anti-chicken IgGH+L coupled to peroxidase (RACh/IgGH+L/PO, Nordic, Tilburg, The Netherlands). In addition, IgM, and IgG antibodies binding to HuSA, respectively, were determined at all sampling times. After incubation with serial dilutions of plasma, plates were incubated with 1:20,000 diluted goat anti-chicken IgM (GACh/IgM) coupled to PO or goat anti-chicken IgGFC coupled to PO, all from ITK Diagnostics, (Uithoorn, The Netherlands). After washing, tetramethylbenzidine and 0.05% H2O2 were added and incubated for 10 min at room temperature. The reaction was stopped with 50 µL of H2SO4. Extinctions were measured with a Multiscan (Lab-systems, Helsinki, Finland) at a wavelength of 450 nm. Titers were expressed as the values (log2) of the dilutions that gave an extinction closest to 50% of the highest mean extinction of a standard positive (pooled) plasma present on every microtiter plate.
Statistical Analysis
Primary total Ab titers to HuSA, primary isotype Ab (IgM and IgG) responses to HuSA, and natural antibody "responses" to RGG, respectively, after primary i.t. PAMP treatment at 7 wk of age were analyzed by a 3-way ANOVA for the effect of breed, treatment (PAMP administration at 7 wk of age), time, and their interactions using the repeated measurement procedure with a "bird nested within (PAMP) treatment" option. Secondary total and isotype Ab responses to HuSA, and primary Ab responses to RGG after the second i.t. challenge with PAMP were analyzed by a 4-way ANOVA for the effect of breed, (first) treatment 1 (PAMP administration at 7 wk of age), (second) treatment 2 (PAMP administration at 13 wk of age), time, and their interactions using the repeated measurement procedure with a bird nested within (PAMP-) treatment 1 and (PAMP-) treatment 2 option. Body weight gain (growth) and FC per time point were analyzed by a 2- or 3-way ANOVA for the effect of breed and first or second PAMP treatment. For feed conversion, primary treatments at 7 wk of age consisted of 6 replicates (cage-breed PAMP treatment). Due to the crossover exposures, only 2 replicates were left after i.t. treatments at 13 wk of age. All analyses were according to SAS Institute (1990) procedures. Mean differences between (PAMP) treatments were tested with Bonferroni’s test.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. For both breeds, and for all PAMP treatments, highest total primary antibody titers to HuSA were found at d 7 postimmunization. In both breeds, titers remained highest at later sampling moments in BGL challenged birds, whereas lowest titers at these sampling moments were found in LPS + BGL challenged birds.
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. The total Ab response (IgT) to HuSA during the complete observation period was significantly affected by a treatment effect (Table 1). Significantly higher total Ab titers to HuSA (P < 0.05) were found in BGL treated birds (treatment 2) as compared with LPS + BGL treated birds (treatment 4) and PBS treated birds (treatment 3), whereas LPS treated birds (treatment 1) were in-between. There were distinct differences between the 2 breeds and the isotype responses to HuSA. White birds had higher IgM Ab titers to HuSA than Brown birds, whereas the opposite was true for IgG Ab titers directed to HuSA. Lipopolysaccharide treatment (treatment 1) significantly decreased IgM Ab titers to HuSA in both breeds, especially in White birds, but BGL (treatment 2) enhanced IgG Ab titers to HuSA. The significant time by treatment by breed interaction for IgM and IgG responses to HuSA was based on the differences between the 2 breeds and enhanced responses in the later periods of the primary responses.
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. Total natural Ab titers binding RGG were affected by breed and by PAMP treatment. Significantly higher natural Ab titers were found in the White birds. 1,3 ß-Glucans (treatment 2) enhanced natural Ab titers binding RGG most pronounced in the White birds at all days after immunization with HuSA (data not shown).
BW (Gain) After i.t. Challenge at 7 wk of Age.
Least squares means of BW and BW gain (
) at 1 and 7 d after i.t. treatment at 7 wk of age is shown in Table 2. Body weight and BW gain were significantly affected by breed; the Brown birds being heavier and growing heavier than the White birds during the observation period. Lipopolysaccharide (treatment 1) and LPS + BGL (treatment 4) significantly decreased BW gain in both breeds as compared with PBS (treatment 3) and BGL (treatment 2). There was no significant breed by treatment interaction.
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. At the week during and 3 wk after i.t. treatments with PAMP, FC was affected by breed and by treatment. At both points, White birds had a higher FC than Brown birds. During the week of i.t. treatment, FC was enhanced in the White birds by LPS (treatment 1) and LPS + BGL (treatment 2). During that period, Brown birds exposed to LPS consumed less feed [on average, 230 g and grew less (15 g), whereas White birds exposed to LPS consumed less (104 g) but also grew less (48 g)], suggesting that in Brown birds, FC was affected by feed uptake, whereas in the White birds, BW loss affected FC. Feed conversion at 3 wk after i.t. exposure was significantly affected by a breed by treatment interaction. At 3 wk after PAMP exposure, FC was decreased in the Brown birds by all PAMP treatments. In that period, FC in Brown birds was still depending on less consumption, whereas in White birds, BW gain affected FC.
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Effects of PAMP Exposure at 13 wk of Age
Exposure of the birds at 13 wk of age was performed to study the following questions. First, are immune-modulating effects of PAMP at 13 wk of age comparable to the effects found at 7 wk of age? Therefore, all birds were immunized with RGG to mount a primary specific Ab response. Second, we measured whether the primary Ab response to RGG, the secondary Ab response to HuSA, and BW gain at 13 wk of age were still affected by the PAMP exposure at 7 wk of age. Thus, some early PAMP-treated birds were challenged with PBS at 13 wk of age. Third, it was studied whether a repeated challenge with the same PAMP at 7 and 13 wk induced a form of adaptation (enhancement or tolerance toward the PAMP) by giving the same PAMP. Fourth, by crossing over PAMP treatments, we studied whether physiological responses depended on early or late PAMP treatment and whether early PAMP treatment resulted in an irreversible modulation of physiological responses. Finally, breed effects were taken into account. Initially, overall statistics are presented; subsequently, results are presented related to the former questions.
Primary Antibody Responses to RGG.
Least squares means of total plasma Ab titers to RGG during 4 wk after primary immunization with RGG at 13 wk of age (and secondary immunization with HuSA) are shown in Table 4.
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). Significantly higher total Ab titers to RGG (P < 0.05) were found in Brown birds. Total primary Ab titers to RGG were not affected by the first i.t. PAMP treatment at 7 wk of age (treatment 1), but the second i.t. PAMP treatment at 13 wk of age (treatment 2) was significant; both LPS and BGL decreased primary Ab responses to RGG. There was a significant interaction between breed and (second) treatment 2. White birds were more sensitive for treatment 2 than Brown birds (i.e., BGL treatment at 13 wk of age decreased total primary Ab responses to RGG as compared with LPS or PBS treatments). There was no significant effect of treatment 1, or an interaction between (first and second) treatments 1 and 2, suggesting that there was no adaptation to repeated or crossover PAMP treatments. Overall, this suggests that with respect to the age of PAMP exposure, birds at 13 wk of age were differently sensitive to PAMP (treatment 2) exposure affecting primary specific Ab responses as compared with 7 wk of age and were not affected by the PAMP exposure at 7 wk of age (anymore).
Secondary Antibody Responses to HuSA.
Total secondary anti-HuSA Ab titers in plasma of birds pretreated i.t. during 5 consecutive days at 13 wk of age with either LPS, BGL, LPS + BGL, or PBS before immunization with HuSA are shown in Figure 2. In both breeds, and in all treatment combinations, highest titers were found at d 7 postimmunization. Depending on the PAMP challenge at 7 wk of age and the PAMP challenge at 13 wk of age, titers were enhanced (LPS or BGL crossover challenges at 7 and 13 wk of age) or decreased (LPS + BGL challenge at 7 wk of age), whereas BGL challenge at 13 wk of age enhanced titers in birds challenged at 7 wk of age with PBS.
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. The total, IgM and IgG Ab responses to HuSA during the complete observation period were significantly affected by breed (Table 4). Significantly higher total Ab and isotype titers to HuSA (P < 0.05) were found in White birds. Total and IgG secondary Ab titers to HuSA were also affected by the first i.t. treatment at 7 wk of age (treatment 1) and the second i.t. treatment at 13 wk of age (treatment 2). There was no interaction between early and late treatments. With respect to treatment 1, significantly lower total Ab and IgG titers were found in the LPS + BGL treated birds of both breeds. After treatment 2, LPS significantly enhanced total Ab titers to HuSA, whereas BGL decreased total Ab responses to HuSA. Secondary IgM titers to HuSA were significantly lower in LPS and BGL treated birds. There were significant interactions among breed, time, treatment 1, and treatment 2.
Prolonged PAMP Effects on Humoral Responses
Prolonged effects of exposure to first PAMP exposure at 7 wk of age on secondary Ab responses to HuSA at 13 wk of age were estimated by contrasts (Table 4) within breeds between first treatment groups 1 (LPS), 2 (BGL), and 4 (LPS + BGL) vs. 3 (PBS) of birds challenged with PBS [i.e., groups 5 (6.09), 11 (5.77), and 23 (4.93) vs. 17 (5.77); Brown birds and groups 6 (6.77), 12 (7.14), and 24 (6.06) vs. 18 (6.42; White birds)]. The combined exposure to LPS + BGL at 7 wk of age significantly depressed total secondary Ab responses to HuSA in both breeds as compared with the birds that either received LPS or BGL.
Comparing the same breed treatment groups, no prolonged effects of early PAMP treatment on secondary IgM and secondary IgG responses to HuSA were found.
In summary, total secondary Ab responses at 13 wk of age were still negatively affected by the LPS + BGL exposure at 7 wk of age, whereas both LPS and BGL exposure at 7 wk of age had a positive effect; however, depending on breed.
Adaptation to Repeated PAMP Exposure Effects on Humoral Responses
To study effects of repeated exposure to the same PAMP at 7 and 13 wk on secondary total Ab and isotype responses to HuSA, we evaluated contrasts (Table 4) within breeds between groups 1 and 2 (LPS-LPS) vs. 9 and 10 (BGL-BGL) vs. 17 and 18 (PBS-PBS) vs. 19 and 20 (LPS + BGL-LPS) and 21 and 22 (LPS + BGL-BGL). With respect to total secondary Ab responses, no contrasts were found in both breeds. With respect to secondary IgM responses to HuSA in Brown birds in groups 1 (3.71),9 (3.35), 17 (3.27), 19 (3.15), and 21 (3.01), no contrasts were found. For White birds in groups 2 (2.89), 10 (3.56), 18 (4.23), 20 (3.41), and 22 (4.15), a significant contrast was found between the LPS-LPS exposed birds (group 2) and the LPS + BGL-LPS exposed birds (group 20) vs. the PBS-PBS treated birds (group 18), suggesting that there was no adaptation in the LPS-LPS or LPS + BGL-LPS exposed birds. With respect to secondary IgG responses, no contrasts were found in both breeds.
In summary, only for secondary IgM responses LPS-LPS and LPS + BGL treatments decreased secondary responses to HuSA in White birds.
Crossover Effects of PAMP on Humoral Responses
To study the effects of exposure at 13 wk of age to another PAMP than the PAMP exposed to at 7 wk of age on secondary total and isotype Ab responses to HuSA, we evaluated contrasts (Table 4) within breeds between groups 1 and 2 (LPS-LPS) vs. 3 and 4 (LPS-BGL) vs. 7 and 8 (BGL-LPS) vs. 9 and 10 (BGL-BGL) vs. 19 and 20 (LPS + BGL-LPS) vs. 21 and 22 (LPS + BGL-BGL). With respect to Brown birds in groups 1 (5.51), 3 (5.88), 7 (6.78), 9 (5.77), 19 (5.65), and 21 (5.04), LPS exposure after BGL exposure induced significantly higher total secondary Ab titers to HuSA, whereas for the White birds, no contrasts were found.
With respect to secondary IgM responses to HuSA, no contrasts were found in Brown birds. In White birds in groups 2 (2.89), 4 (3.24), 8 (3.97), 10 (3.56), 20 (3.41), and 22 (4.15), exposure to BGL at 13 wk of age after exposure to LPS + BGL at 7 wk of age induced significantly higher secondary IgM titers to HuSA.
With respect to secondary IgG responses to HuSA in Brown birds in groups 1 (2.73), 3 (2.93), 7 (3.60), 9 (2.80), 19 (2.61), and 21 (2.27), an LPS exposure at 13 wk of age after BGL exposure at 7 wk of age induced higher secondary IgG titers to HuSA. In White birds, no contrasts were found.
In summary, the present data suggests that crossover exposure, depending on breeds, enhanced secondary total and isotype Ab responses to HuSA.
BW (Gain) After i.t. Challenge at 13 wk of Age.
Least squares means of mean BW and BW gain () at 1, 8, and 28 d after i.t. treatment at 13 wk of age is shown in Table 5. Body weight and BW gain were significantly affected by breed, the Brown birds at all sampling times being heavier and growing faster than the White birds during the observation period. Body weight gain of both lines at 1 d after PAMP exposure at 13 wk of age was significantly affected by second treatment 2. Lipopolysaccharide exposure significantly decreased BW gain. However, with respect to first treatment 1 at 7 wks of age, birds that had received LPS showed lower BW loss when challenged at 7 wk of age with LPS as compared with PBS challenged birds. At 1, 8, and 28 d after secondary exposure at 13 wk of age, there was a significant treatment 1 by treatment 2 interaction. Birds that had received LPS or LPS + BGL at 7 wk of age (treatment 1) and were subsequently exposed to LPS at 13 wk of age (treatment 2) showed higher BW gain or less BW gain "loss" than other treatments. This was true for both lines at d 1 and 8 and for Brown birds only at d 28.
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1), 8 d (8), and 28 d (28) after exposure were estimated by contrasts (Table 5) within breeds between early treatment groups 1 (LPS), 2 (BGL), and 4 (LPS + BGL) vs. 3 (PBS) of birds challenged at 13 wk of age with PBS (i.e., groups 5, 11, and 23 vs. 17 for Brown birds and groups 6, 12, and 24 vs. 18 for White birds). With respect to 1 in Brown birds, groups 5 (5), 11 (16), and 23 (–1) vs. 17 (3) revealed that first BGL exposure (group 11) resulted in a significantly higher 1 as compared with the other PAMP exposures, including PBS at 7 wk. In White birds, groups 6 (2), 12 (14), 18 (1), and 24 (9) also revealed an enhancing effect of BGL exposure at 7 wk of age (group 12). With respect to 8 and 28, there were no prolonged effects of first PAMP exposure in both breeds.
Adaptation to Repeated PAMP Exposure Effects on BW Gain
To study effects of repeated exposure to the same PAMP at 7 and 13 wk on BW gain:1, 8, and 28, we evaluated contrasts (Table 5) within breeds between groups 1 and 2 (LPS-LPS) vs. 9 and 10 (BGL-BGL) vs. 17 and 18 (PBS-PBS) vs. 19 and 20 (LPS + BGL-LPS) and 21 and 22 (LPS + BGL-BGL). With respect to 1 in Brown birds in groups 1 (–16), 9 (12), 17 (3), 19 (1), and 21 (5), repeated exposure to LPS (group 1 and, to a minor extent, group 19) showed the lowest BW gain. In White birds, groups 2 (18), 10 (12), 18 (1), 20 (–2), and 22 (9) revealed that repeated exposure to LPS at 13 wk of age resulted in a lower 1 after early exposure to LPS + BGL (group 20). With respect to 8 in Brown birds in groups 1 (68), 9 (28), 17 (25), 19 (50), and 21 (38), repeated exposure to LPS (group 1 and, to a minor extent, group 19) showed the highest BW gain. In White birds, groups 2 (62), 10 (24), 18 (22), 20 (51), and 22 (29) revealed that repeated exposure to LPS at 13 wk of age resulted in a higher 8 after early exposure to LPS (group 2) or LPS + BGL (group 20). With respect to 28 in Brown birds in groups 1 (402), 9 (299), 17 (334), 19 (339), and 21 (323), repeated exposure to LPS (group 1 and, to a minor extent, group 19) showed the highest BW gain. In White birds, groups 2 (305), 10 (283), 18 (254), 20 (316), and 22 (303) revealed that repeated exposure to LPS at 13 wk of age after early exposure to LPS + BGL (group 20) resulted in the highest BW gain.
Crossover Effects of PAMP on BW Gain
To study the effects of exposure at 13 wk of age to another PAMP than at 7 wk of age on BW gain, we evaluated contrasts (Table 5) within breeds between groups 1 and 2 (LPS-LPS) vs. 3 and 4 (LPS-BGL) vs. 7 and 8 (BGL-LPS) vs. 9 and 10 (BGL-BGL) vs. 19 and 20 (LPS + BGL-LPS) vs. 21 and 22 (LPS + BGL-BGL). With respect to 1 in Brown birds in groups 1 (–16), 3 (17), 7 (–28), 9 (12), 19 (1), and 21 (5), birds that received LPS, irregardless of the PAMP they received at 7 wk of age, showed decreased BW gain. For White birds, groups 2 (18), 4 (10), 8 (2), 10 (12), 20 (–2), and 22 (9) showed that birds that were exposed to LPS at 13 wk following earlier exposure to LPS at 7 wk of age (group 2) "suffered" less BW loss than birds that were exposed to the other PAMP at 7 wk of age. With respect to 8 in Brown birds in groups 1 (68), 3 (42), 7 (36), 9 (28), 19 (50), and 21 (38), birds that were exposed to LPS at 7 wk of age (group 1) showed higher BW gain at 8 d after exposure to LPS at 13 wk of age. For White birds in groups 2 (62), 4 (46), 8 (31), 10 (24), 20 (51), and 22 (29), birds that were exposed to LPS or LPS + BGL at 7 wk of age had higher BW gain at d 8 after exposure to LPS at 13 wk of age than the other combinations of exposure.
With respect to 28 in Brown birds in groups 1 (402), 3 (347), 7 (296), 9 (299), 19 (339), and 21 (323), birds that received LPS at 7 wk of age showed higher BW gain at 13 wk of age when exposed to LPS at 7 wk of age (group 1). For White birds, there were no contrasts, but birds that were exposed to LPS + BGL at 7 wk of age had the highest BW gain at d 28 after exposure to LPS at 13 wk of age.
FC After i.t. Challenge at 13 wk of Age.
No significant effects of breed, treatment 1, treatment 2, or their interactions on FC were found after i.t. treatments at 13 wk of age (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We addressed various questions. First, it was studied what PAMP at what age affected primary and secondary immune responses (to HuSA and RGG). Therefore, primary immune responses were measured at 2 ages. Earlier, we found that LPS, when administered i.v., enhanced in vitro cellular immunity but decreased primary Ab responses (Parmentier et al., 2004) for several months, suggesting that LPS activates a TH-1–like route (cellular response) and may polarize immune responses to other antigens in this direction (Kapsenberg, 2003). In the present study, however, although LPS decreased primary IgM antibody responses, LPS enhanced secondary humoral (IgG) responses, as found earlier (Maldonado et al., 2005). Lipopolysaccharide was reported to induce more interleukin-10 (IL-10) than IL-12 production in vitro, though less-than-intact gram-negative bacteria did (Hessle et al., 2000). IL-12 stimulates T and NK cells to secrete interferon- and to lyse target cells, but IL-10 stimulates B-cell maturation and antibody production (Hessle et al., 2000). Our data suggest that in poultry LPS can also polarize secondary immune responses to the TH-2 route. It is noteworthy that exposure of mice to LPS may result in an enhanced production of IL-10 as a regulatory response to the LPS induced production of IL-12 (Varma et al., 2005). This suggests that exposure to LPS can result in different pathways of cytokine production and, as a consequence, disease resistance, depending on age of the bird, intensity of challenge, or both. The present results show that both BGL and LPS have immune-modulating features, which may depend, however, on the route of challenge, dose, and duration of challenge. In general, BGL enhanced both natural (RGG), and specific primary at 7 wk and secondary Ab responses to HuSA at 13 wk of age but did not enhance specific primary Ab responses (to RGG) at the latter age. A combination of LPS + BGL negatively affected secondary antibody responses to HuSA. The latter suggested that exposure at 7 wk of age still had a prolonged effect on humoral responsiveness. Effects of i.t. administration were pronounced with respect to secondary IgG responses. This was in agreement with earlier findings (Maldonado et al., 2005) and is not in contradiction with the hypothesis that PAMP like LPS or BGL also directly activate memory B cells via binding to TLR, maintaining a nonantigen specific form of memory (Bernasconi et al., 2003). Our data suggest that birds were more sensitive for immune modulation at 7 wk of age as compared with 13 wk of age. In addition, the early treatment still affected responses at 13 wk of age, although not irreversibly.
Analyses of contrasts within breed and treatment groups not only revealed breed and treatment effects but also indicated that secondary immune responsiveness was enhanced by crossover exposures, whereas loss of BW gain after exposure of 13 wk of age was less in birds that were repeatedly exposed to the same PAMP, suggesting differences between the immune system and growth in the adaptation to hygienic conditions. Lipopolysaccharide, but not BGL, affected BW (gain), as a parameter of cachectin activity, when administered at 7 wk, in a dose-dependent fashion. However, birds, when treated at 13 wk of age with LPS, showed less cachectin responses when exposed to LPS, BGL, or LPS + BGL at 7 wk of age, as compared with birds that were exposed for the first time to LPS at 13 wk. This suggests that the earlier LPS challenged birds adapted to the LPS (i.e., hygienic conditions) at 7 wk of age. Also at d 7 and 28 after secondary exposure with PAMP, highest BW gain was found in the birds that had been exposed to LPS or LPS + BGL at 7 wk of age and, in addition, were challenged with LPS at 13 wk of age again. Previously, it was shown that repeated LPS injections may cause broiler chicks to become refractory to the LPS stimulus (Korver et al., 1998). Neonatal rats challenged with LPS showed suppressed febrile responses and reduced NF-
ß activation in their lymphoid organs but enhanced corticosterone responses when challenged with LPS later in life, suggesting that their innate immunity is programmed by neonatal LPS challenge (Ellis et al., 2005). Similarly, it was shown that tolerance to LPS is an active process including enhanced expression of NF-ß, TNF receptor type II, and IL-10, which are involved in the downregulation of proinflammatory cytokines (Ziegler-Heitbrock, 1995). In the current study, administration of BGL or BGL + LPS also reduced cachectin responses toward LPS at 13 wk. This suggests a non-PAMP specific adaptation to hygienic conditions early in life. On the other hand, we found enhanced secondary Ab responses to HuSA in those birds that had undergone crossover PAMP treatments, which suggests that activity of the humoral immune system is enhanced by different PAMP stimuli.
Taken together, the present results show that immune modulation by airborne PAMP is possible, but the effects depend on breed, age of the bird, and earlier (combinations of) exposure. Regardless of the nature of exposure at an early age, antibody responses were affected at a later age. Chickens, however, adapted in a breed-dependent fashion to exposure at 7 wk of age, which was reflected by the effects of this exposure on immune reactivity and BW gain at 13 wk of age. Tracheal challenge may reflect the hygiene status of a chicken house, although it remains to be established whether continuous ("low dose") exposure or temporary ("high dose") exposure underlie modulation of the immune response or growth. Modulation of the immune reactivity by airborne PAMP, either intentionally or not, may have serious consequences for the health status of an individual animal (Sainsbury, 1992; Appleby et al., 2004). Not only do animals kept under clean hygienic conditions grow faster and produce better, continuous exposure to airborne endotoxins has been related with (human) health disorders [e.g., respiratory (hypersensitivity) diseases in farmers (Douwes, 1998, Douwes et al., 2004)] and may also affect the health of poultry (Powers et al., 2005). Humans with chronic heart failure showed cellular hypersensitivity to LPS and a higher capacity for TNF
generation (Krüger et al., 2004). Long term i.t. exposure to LPS of mice resulted in production of proinflammatory cytokines, persistent pathology, and chronic lung inflammation in mice (Ishii et al., 1997; Vernooy et al., 2002) and increased pulmonary arterial pressure in broilers (Wideman et al., 2004; Chapman et al., 2005), most of which are, however, acute reactions.
High antibody responsiveness could add to a better resistance to certain diseases; however, polarization of the immune response could also result in unwanted hypersensitivity or allergy, or decreased resistance to other infections. Finally, the application of vaccination schemes and hygienic conditions in chicken houses could interfere. In various housing systems (organic, biological), birds will undergo PAMP challenge in a variable fashion [i.e., in a variety of (aerial) environments: inside (high level), outside (low level)] as well as undergo different (temporary) exposures to levels of LPS (straw and organic material in stables). Furthermore, preventive administration of antibiotics will be discouraged, whereas food animals will progressively encounter other pathogens. We suggest that the continuous presence of airborne PAMP in stables might not only be related with respiratory disorders, suboptimal health, and (re)production but also affect the way by which birds respond to infection. This necessitates further knowledge of the effects of airborne components on the immune system, especially at very early stages of life, and more knowledge of the "life history" of birds, not only to understand variations in immune responses but also to consider the reported endotoxin affected corticosterone levels and, consequently, implications for stress-related diseases in elder rats (Shanks et al., 2000; Shanks and Lightman, 2001). Study of the mechanistic basis underlying the proposed deleterious but, on the other hand, possible advantageous effects of LPS and other PAMP such as ß-glucans or CpG DNA on the health of birds should add to more optimal husbandry management of different breeds.
Received for publication May 8, 2006. Accepted for publication July 12, 2006.
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), 1,3 ß-glucan (BGL; 
), PBS (
), or LPS + BGL (•) at 7 wk of age during 5 consecutive days before s.c. immunization with 1 mg of HuSA.