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Keywords:

  • immunological imprinting;
  • maternal effects;
  • yolk antibodies

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

1. Immunological imprinting by maternally derived antibodies has been proposed to have both positive and negative consequences for offspring immunity in early and adult life. However, few studies of maternal effects on immunity have followed individuals past the juvenile stages.

2. Using laboratory Japanese quail, we developed a novel method of directly manipulating yolk antibodies of neonates, and then followed individuals through a series of immune challenges until they were of reproductive age.

3. Our method of directly injecting purified antibodies into the yolk sac of newly hatched chicks successfully elevated the plasma titres of specific anti-KLH IgY in neonates. This allows us to test whether differences in neonatal anti-KLH IgY affect immunity at the juvenile and adult stages of life.

4. We found little evidence for an effect of maternal antibodies on juvenile stage immune response, in contrast to results from previous studies. Adult immune response depended largely on the magnitude of the juvenile immune response regardless of the identity of the antigen in the juvenile immune challenge, and did not depend on neonatal IgY titres. Our results are consistent with a priming effect of early immune experience on adult stage immune responsiveness, but we found no evidence of carryover effects of yolk-derived antibodies on adult immunity.

5. This study employs new methodology for investigation of maternal antibodies and presents results suggesting that further studies of maternal effects on immunity will require careful consideration of the numerous ways maternally derived yolk components can impact the different types of immune response.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Maternally derived antibodies have been shown to benefit neonates by markedly improving immunity (Tizard 2002; Grindstaff, Brodie & Ketterson 2003), reducing acute phase responses to challenge (Goldyne 2000), and increasing growth rate (Grindstaff & Ketterson 2001; Pihlaja, Siitari & Alatalo 2006; Grindstaff 2008). Moreover, the nutritional demands of maternal antibody allocation in domestic poultry have been shown to be negligible (Klasing 1998; Grindstaff, Demas & Ketterson 2005), though costs remain relatively unstudied in wild systems. Given these substantial benefits, the observed levels of maternally derived antibodies in avian eggs are remarkably low.

Potential indirect costs of maternal antibody provisioning include compromised offspring immune development (Carlier & Truyens 1995) and autoimmune disorders triggered by maternal antibodies (Greeley et al. 2002; Von Herrath & Bach 2002; Lemke et al. 2009). Environmental and self-antigen exposure is essential to the diversification and development of B cells (Baumgarth, Tung & Herzenberg 2005), influencing the maturation and refinement of natural immunity (Apanius 1998). Maternally derived antibody circulating in neonates binds antigens introduced via infection or environmental exposure, masking those antigens from the developing B cells, and possibly preventing positive selection for matched variable-region B cells. The resulting cost could be two-fold: offspring may develop a less diverse set of B cells due to a reduction in positive selection, and they may have reduced resistance to specific infectious agents upon re-exposure later in life.

Early immune experience has lasting effects on the immune response, through the production of memory cells (Tizard 2002) or desensitization to antigens (Wills-Karp, Santeliz & Karp 2001). Exposure to some antigens early in life provides lifetime immunity against those pathogens. Vaccination scheduling is designed to take advantage of this feature of the immune system, while taking into account potential interference of maternal antibodies (Pastoret 2007). Despite an extensive literature on maternal antibodies and vaccination schedules, individuals are seldom followed past the juvenile stage to reproductive age, and so little is known about whether maternal antibodies influence the adult immune response of birds.

We initiated this study to determine whether high concentrations of maternally derived antibodies in neonates could depress the immune response later in life. This study investigates longer-term impacts of maternal antibodies on the specific adaptive arm immune responses in offspring of the precocial domestic Japanese quail (Coturnix coturnix japonica). We generated a harvestable pool of yolk antibodies (IgY) by hypervaccinating hens against one of two well defined challenge antigens: bacterial lipopolysaccharide (LPS) or dendroaspis natriuretic peptide conjugate – keyhole limpet hemocyanin (KLH). These antigens represent both T-cell independent (LPS) and T-cell dependent (KLH) antigens, thus presenting the opportunity to assess the impacts of maternal IgY on both types of immune response. Harvested IgY was then used to manipulate yolk IgY levels of newly hatched chicks, which were subsequently given a series of challenges as juveniles and adults to test immune competency against these antigens. We predicted that yolk IgY treatment would diminish the IgY response at both the juvenile and adult stages in a dose-dependent fashion.

Materials and methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Harvest of quail yolk IgY

We created two IgY types by harvesting yolk IgY from quail hens vaccinated with either LPS (from Salmonella enterica typhimurium; Sigma, St Louis, MO, USA) or KLH (CalBioChem, San Diego, CA, USA). Eight hens for each antigen received four vaccinations, spaced every 3 days, with either LPS or KLH (1 mg kg−1) antigen in a 50 : 50 mixture with Freund’s incomplete adjuvant (Sigma) to promote B-cell responses. One week after the first vaccination when antibody response is peaking (Tizard 2002), we started to collect eggs, which we continued for 2 weeks to harvest sufficient IgY for our yolk injection treatments. Yolk IgY was purified by two-step PEG-6000 (polyethylene glycol; Fluka Chemical, St Louis, MO, USA) extraction [a modification of the Polson method outlined in Stålberg & Larsson (2001)]. First, yolk was measured into a conical centrifuge tube and four volumes of 3·5% PEG in saline was added, and thoroughly vortexed. We incubated the solution at 4 °C for 4 h, and then centrifuged at 5000 g for 30 min at 4 °C. The supernatant was reserved in a new conical tube, and the lipid discarded. One volume of 24% PEG in saline was added to the supernatant, and the solution was vortexed and incubated at 4 °C for 1 h. The solution was again centrifuged at 5000 g for 30 min at 4 °C, the supernatant poured off and the white IgY pellet reserved. The IgY pellet was briefly rinsed with −20 °C ethanol to remove residual PEG, and dried in a fume hood. The dry precipitate of all eggs in each antigen treatment was pooled, and the extraction process repeated to further purify the IgY. Dry IgY from all eggs in each treatment was weighed, and rehydrated in saline at a concentration of 800 mg mL−1, and stored at 4 °C. We checked total IgY concentrations of stock solutions by direct ELISA using a chicken IgY (Sigma) standard curve.

Effects of yolk IgY on humoral immune development

We tested the effect of yolk IgY treatment in a complete block design using siblings matched across treatments (Table 1). Our experiment had four treatment combinations: injection of 20 mg albumin (control), 2 mg anti-KLH IgY with 18 mg albumin resulting in an equivalent protein level (low KLH), 20 mg anti-KLH IgY (high KLH) and 20 mg anti-LPS IgY (high LPS). Note that our treatments are not monoclonal antibodies, but a mixture of antibodies deposited into eggs after vaccination of the hens. Injections of 0·05 mL volume protein dissolved in sterile saline were made into the yolk sac of newly hatched chicks using 0·5 cc tuberculin syringes with 27 g needles. The treatment levels of yolk IgY were selected to reflect near natural levels of IgY in quail eggs from unvaccinated hens [about 2–4 mg, unpublished data and (Grindstaff, Demas & Ketterson 2005), bringing yolk IgY levels to 4–6 mg for chicks in the low KLH group] and levels well outside of the vaccinated range [of 10 mg (Grindstaff, Demas & Ketterson 2005), bringing yolk IgY levels to about 22–24 mg for high KLH and LPS groups, more on this in results]. Small blood samples (50 μL) were drawn at 2 days of age for ELISA confirmation of IgY incorporation into circulation (neonatal IgY titres). At 7 days of age, half of the sibling sets were challenged with LPS and the other half with KLH (juvenile stage challenge). Blood was sampled again 10 days post-challenge for ELISA measurement of anti-LPS or anti-KLH antibodies (juvenile IgY titres). All birds were challenged again with KLH or LPS in alternate challenges at five and 6 weeks of age (adult stage challenge, Table 1), and blood was sampled for IgY response 10 days later (adult IgY titres). All challenge doses were 1 mg kg−1 of body weight administered in solution concentrations adjusted to approximately 0·05 mL vaccination volumes injected subcutaneously over the shoulders. All birds were weighed at the time of vaccination and injection volumes adjusted slightly to administer uniform antigen dosages across all individuals.

Table 1.   Experimental design with four treatments and four challenge schedules using sibling sets from 46 hens
Challenge scheduleTreatments
1 week5 week6 weekControl albuminHigh IgY (anti-LPS)Low IgY (anti-KLH)High IgY (anti-KLH)
  1. The numbers represent the n value for the particular treatment*challenge block. There are 12 sibling sets for each challenge schedule, with one sib from each set in each treatment (some sibling sets were incomplete due to hatching failure or death), and a total experimental n of 160. Chicks from the anti-LPS IgY yolk treatment group were excluded from subsequent analyses of juvenile and adult immunity.

LPSKLHLPS612109
LPSLPSKLH8101011
KLHKLHLPS1011109
KLHLPSKLH11111111

Serum samples were stored at −20 °C until ELISA analysis. Antibody titres were measured by sandwich ELISA as in Hasselquist et al. (1999) by binding 2μg mL−1 solutions of LPS or KLH antigens in carbonate-bicarbonate binding buffer to 96-well plates, blocking with 1% bovine serum albumin (BSA; Sigma), adding 100 μL of 1 : 100 dilutions of plasma in PBS-tween, blocking with 1% BSA, and adding a 1 : 20 000 dilution of HRPO-anti-bird IgY (Bethyl Laboratories, Montgomery, TX, USA) secondary antibody in blocking buffer. Colorimetric detection employed TMB PO substrate (Thermo Scientific, Rockford, IL, USA) stopped with 1N sulphuric acid after 15 min, and the plate was read at 450 nm. A standard was run using serial dilutions of pooled serum samples from all birds collected after the second immune challenge. Samples were run in duplicate and duplicates with CVs greater than 0·15 were rerun and the outlier discarded. All IgY values are from the averaged duplicates and expressed as titres relative to the pooled serum standard and then log transformed; thus each sample titre is a log proportion of the pooled serum IgY concentration.

Statistical analysis

Data were analysed in three steps using AICc model selection and goodness of fit tests (Burnham & Anderson 2002). First, we determined the efficacy of the yolk sac injection treatments. Second, we assessed whether yolk derived antibodies affected the response to challenge as a juvenile. Third, we assessed whether yolk derived antibodies or juvenile challenge antigen affected the adult stage response. All models included sibling group as a random effect and were estimated using the lmer() function in the lme4 package of R (v. 2.7.2, The R Foundation, Vienna, Austria).

The efficacy of yolk sac injections was evaluated by simple AICc comparison of mixed models for differences between groups against a null model. Separate analyses were conducted for anti-KLH and anti-LPS IgY titres. The anti-LPS IgY manipulations were not effective (see Results) and so all subsequent analyses of the anti-LPS yolk treatment groups were excluded.

To assess the effects of yolk derived IgY and challenge antigen on juvenile response, we compared three models including a null model, a model for yolk treatment, and a model for neonatal IgY titres. If yolk derived antibodies affected juvenile response we would expect the models including yolk treatment and neonatal antibody titres to assume the highest weight, and the null the lowest weight.

To assess the effects on adult stage response we compared eight models including a null model, which included the terms yolk treatment, neonatal antibody titre, juvenile challenge antigen, vaccination schedule, the magnitude of juvenile response (to KLH or LPS challenge) and interaction terms. The responses of the week 5 and week 6 challenges were pooled as ‘adult response’, which is why the vaccination schedule term was included among the models. The model hypotheses are outlined in Table 2.

Table 2.   Models for analysis of adult KLH and LPS responses Thumbnail image of

All models were assessed for goodness of fit using the rescaled generalized coefficient of determination (R2) definition from Nagelkerke (1991) which utilizes likelihoods of the model of interest and the null model.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Yolk injection treatments

The anti-KLH titres differed among yolk treatment groups (ΔAICc = 28 for the null model, AICcw∼1 for the treatment model, R2 = 0·91, Fig. 1a). However anti-LPS titres did not differ among yolk treatment groups (ΔAICc = 10 for the treatment model, AICcw∼1 for the null model, R2 = −0·10, Fig. 1b). The absolute difference among treatments was not as large as would be expected given the differences in yolk sac injection amounts, anti-KLH titres were about 1·5× control for the low-KLH treatment, and 3× control for the high KLH treatment, and anti-LPS titres were about 2× control for the high LPS treatment.

image

Figure 1. (a) Neonatal anti-KLH IgY differed among yolk treatment groups (mean ± 95% CI), indicating that yolk sac IgY injections were incorporated into circulation. (b) Neonatal anti-LPS IgY did not differ among treatment groups.

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Juvenile response

The juvenile stage response level was not explained by yolk treatment (Table 3). Neonatal anti-KLH IgY titres ranked above yolk sac treatment group for predicting juvenile response, but still have relatively low AICc weight. Because of the weak explanatory power of the models we do not report effect size, as the confidence intervals include zero. Thus, yolk antibodies have no measureable effect on antibody response at the juvenile stage.

Table 3.   AICc model selection tables for juvenile antibody responses to KLH challenge
Juvenile KLH responsenKlogLAICcΔAICcLΔAICcAICcWR2
  1. All models include sibling group as a random effect on intercept.

Null453−29·64365·8720·0001·0000·7830·000
Neonatal anti-KLH titre454−29·76168·5232·6510·2660·208−0·007
Yolk sac treatment455−31·67974·8969·0240·0110·009−0·129

Adult response

For adult stage KLH response the best-fit model was the juvenile response alone, independent of juvenile antigen exposure; models including juvenile antigen exposure ranked relatively high with ΔAICc values in the range of plausible hypotheses with ΔAICc values less than 10 (Table 4). However, model fit for the juvenile response effect was poor, with R2 = 0·101 (Fig. 2).

Table 4.   AICc model selection for response to vaccination at the adult stage
Adult KLH responsenKlogLAICcΔAICcLΔAICcAICcWR2
  1. Early immune response, independent of the antigens involved, best predicted the adult antibody response to vaccination with KLH. Sibling group was used as a random effect on intercept.

Early immune response/pleiotropy784−69·452147·4480·0001·0000·7680·101
Null783−72·937152·1994·7510·0930·0710·000
Antigen familiarity784−71·957152·4615·0130·0820·0630·029
Neonatal specific antibody titre784−72·307153·1625·7140·0570·0440·019
Early immune response depending on antigen familiarity786−69·994153·1835·7350·0570·0440·086
Yolk derived antibodies (treatment)785−73·170157·1749·7260·0080·006−0·007
Age of vaccination (vaccination order)786−72·467158·11710·6690·0050·0040·014
Yolk derived antibodies and immune experience7814−65·454165·57418·1260·0000·0000·206
image

Figure 2. Juvenile challenge response had a positive effect on adult response to KLH, regardless of the antigen in the first vaccine, and the order of subsequent vaccines. Circles are challenge schedule KLH-KLH-LPS, squares are KLH-LPS-KLH, upward triangles are LPS-KLH-LPS, and downward triangles are LPS-LPS-KLH.

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Using egg extracted antibodies to manipulate neonatal antibody titres through yolk sac injections worked well for anti-KLH antibodies, but was relatively ineffective for anti-LPS antibodies. There are likely two reasons for this. First, LPS is a general B cell mitogen and hen vaccination with LPS may have stimulated B cells not specific to LPS. Second, the KLH vaccination may likewise have stimulated overall antibody production (to a lesser degree than LPS) in addition to the specific anti-KLH B cell responses. Thus, we would recommend against the use of LPS for future investigations of specific responses.

Yolk sac injections of newly hatched chicks have not previously been used in ecological research to study the effects of maternal antibody allocation (Boulinier & Staszewski 2008). However, this is a useful technique for manipulating neonatal IgY because it allows more precise control over the maternal effect compared to maternal vaccination, which may alter inflammation (Tizard 2002) and subsequently steroid hormone levels (Sapolsky, Rivier & Yamamoto 1987) in addition to yolk IgY. Not all antibody injected into the yolk sac was absorbed into circulation; neonatal anti-KLH antibodies were not 10× higher in the high treatment level compared to the low treatment level. This could be because the Fc receptors on chick yolk sac were saturated at a level between our high and low treatment levels. We have no information about the fate of antibodies not appearing in circulation but it is likely that they would be deposited into the gut with unabsorbed yolk. The use of ovalbumin as a sham is a reasonable control for nutritional effects of yolk IgY, and this might become more important if injection treatment concentrations were significantly increased. The major challenge with this technique is to obtain antibodies that will be absorbed and function as maternally derived antibodies for the species of interest. For studies in wild populations, it may be possible to use antibodies from donor eggs from the same population, or to explore whether commercially available bird IgY can be used in other species.

In contrast to previous studies on maternally derived antibodies, we found no effect of yolk antibodies on the juvenile immune response. Previous studies have reported both positive and negative effects of yolk antibodies on antigen-specific antibody production in chicks (Hassan & Curtiss 1996; Grindstaff et al. 2006; Reid et al. 2006; Staszewski et al. 2007). There are several possible explanations for a lack of effect. Manipulated levels of yolk antibodies were within a range of physiologically relevant concentrations. Vaccination doses were probably insufficient to overcome the yolk antibody levels because the vaccine dosage selected was the minimum likely to cause a B cell response (Koutsos & Klasing 2001). Alternatively, chicks might respond to the particular antigens utilized through a route not affected by maternal antibodies. Current ideas about the mechanism of maternal antibody action are that the antibodies block epitopes from being recognized by immune cell receptors, however this may not be a complete picture of maternal effects on immunity. The time window within which maternal antibodies affect immunity could occur prior to our yolk sac manipulation during pre-hatching uptake. Yolk antibody uptake starts early but very slowly and increases exponentially 2 or 3 days prior to hatch, continuing for about 2 days post-hatch in chickens (Kowalczyk et al. 1985). However, a recent study (Abou Elazab et al. 2009) that used a similar, but in ovo, manipulation of yolk antibodies found strong effects of yolk derived anti-KLH antibodies on offspring immune response to KLH. This study used much higher levels of affinity-purified anti-KLH antibodies for manipulation, suggesting that these do have a potential blocking effect on offspring response, but this fails to test effects within the natural range, and in the context of maternal anti-idiotypes that may also play an important role in offspring immune development (Lemke, Coutinho & Lange 2004).

The lack of a strong effect in our yolk antibody manipulations may also imply that synergistic effects of other changes involved with vaccination may be more important in the response suppression normally associated with maternal antibodies. Previous studies of maternal antibodies have manipulated offspring levels via vaccination of the mother (Reid et al. 2006; Staszewski et al. 2007). Relatively little attention has been paid to effects on other egg components after maternal vaccination; this may be an important direction for future research. Additionally, there has been no attempt to distinguish between types of antigens and whether the effects of maternal antibodies could differ depending on the pathogen of interest, despite clear differences in neonatal immunity against different antigens (Siegrist 2007).

It appears from calculations of the nutritional cost of yolk antibody allocation that this is not what limits yolk antibody levels in large bodied domestic birds (Klasing 1998). A recent phylogenetic analysis of yolk antibody levels suggests a developmental constraint on yolk antibody levels (Addison et al. 2009), which may be greater than the nutritional constraints indicated by relationships with body size. If the nature of that constraint is in the development of the specific immune response, the evidence so far is equivocal. Future studies should focus on determining whether there are certain types of antigens for which maternal antibodies are beneficial or detrimental. It would also be useful to use an experimental design that utilizes yolk injections of antibodies to determine the exact timing of action of maternal antibodies on offspring immune function.

We found evidence for a positive relationship between juvenile response and adult response, regardless of whether the antigen of the adult vaccination matched the juvenile vaccination. This effect is not due to memory cell production because it was independent of the juvenile challenge antigen. It is likely due to a combination of factors including a generalized effect of early immune experience on development and subsequent reactivity of leucocytes. We partially controlled for genetic factors by including sibling group as a random effect on intercept in our models, suggesting that pleiotropic effects may not be the only factor contributing to this relationship. Some evidence suggests that genetic factors play a more important role in juvenile immune response than in adult immune response (Kimman, Vandebriel & Hoebee 2007), so this pattern might arise not from direct genetic factors but indirectly via carryover effects from the juvenile challenge response.

We found no evidence of carry over effects of maternal IgY into adult immunity, at least when yolk antibody levels approached high vaccine response levels. However, yolk antibodies had little or no effect on juvenile immune response, and so it is not surprising that there were no long-lasting effects. Thus, we found no evidence that the costs of maternal antibody allocation are borne in the specific immune response. The ecoimmunology literature has so far addressed only the adaptive significance of yolk antibodies, and the costs remain largely unknown (Hasselquist & Nilsson 2009). Potential developmental effects of yolk antibodies such as diversification of the B cell repertoire, or autoimmunity, remain unstudied. Our study presents a novel technique for manipulating yolk antibodies, and this can now be utilized to test other hypothesized effects of maternally derived antibodies on offspring development.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

We would like to thank members of the Klasing lab, especially Laura Flatow, Edgar Garcia, Kim Livingston and Arash Naziripour for their help throughout this study, and Kristy Smith and Jackie Pisenti in the avian facilities at UC Davis. Funding for this research came from NSF grant IBN-0212587 to RER and KCK, a GIAR from the Society for Integrative and Comparative Biology to BA, and an NSERC PGS D to BA. This study was done under IACUC protocol # 05-11972 at UC Davis. The manuscript benefited greatly from the constructive comments from two anonymous reviewers.

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  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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