Introduction

The adaptive humoral (or ‘antibody-mediated’) immune system of birds comprises a highly protective, specific arm of defense that retains long-term memory of invading pathogens [1], [2]. The model for avian humoral immune function proposes that production of adequate numbers of peripheral B-cell lineages that express functionally different immunoglobulin (Ig) specificities requires at least four to six weeks to develop in all birds [3]. This model is derived from studies of precocial galliformes, in which B-cell development begins embryonically during approximately the final week of egg incubation and is completed approximately three to six weeks after hatching, well before chickens reach adult size [2]–[4]. However, some altricial bird species, which have short incubation times and among the fastest growth rates of land animals [5], [6], fledge at adult size less than four weeks after the start of embryonic development. Given the proposed model of humoral immune development, and evidence that maternal antibodies (Igs) from the egg are catabolized within two weeks post-hatch in altricial birds [7]–[9], it is likely that rapidly-developing altricial birds are immunologically immature and relatively vulnerable to infection throughout the nestling and early post-fledging periods.

Non-infectious antigen vaccinations and antigen-specific immune assays have been used to examine induced antibody responses in developing birds. Upon first exposure to an antigen, a primary antibody response predominated by IgM is generated, isotype class-switching occurs, and memory B-cells are stored. Upon repeated exposure to the same antigen, a rapid secondary, memory response occurs, predominantly in the form of IgY antibodies [1], [2]. Capacity for specific primary antibody response was detectable in altricial pigeons, pied flycatchers, and American kestrels injected with foreign antigen during the first week post-hatch [10]–[12]. Primary response levels were been shown to increase and become more reliable after the first week post-hatch in altricial pigeons and macaws [10], [13], which was in agreement with studies of response levels in precocial chickens and turkeys [14]–[16].

Fewer studies have examined the ontogeny of secondary, adaptive antibody responses in altricial birds. Pigeons initially injected with sheep red blood cells (SRBC) during the first 10 days post-hatch produced IgY antibodies when a second immunization was administered 2–3 weeks later, indicating that the capacity for class-switching from IgM to IgY and memory cell formation occurs within the first weeks of hatching. Secondary antibody responses to repeated injections of SRBC and the non-pathogenic conjugate dinitrophenol keyhole limpet hemocyanin (DNP-KLH) were studied in American kestrels using hemagglutination and ELISA assays, respectively [17]. Kestrels first injected at 10 days post-hatch and boosted with the same antigen 6 days later showed robust secondary antibody responses, though detection of responses to DNP-KLH using ELISA was more sensitive than the SRBC hemagglutination assay [17]. More detailed studies of age-related changes in adaptive secondary antibody response of kestrels found significantly higher secondary responses in birds first vaccinated with DNP-KLH at 7–9 days post-hatch compared with those vaccinated at 3–5 days post hatch, and that adult secondary response was four times higher than that of nestlings [12]. Based on these data, it is likely that altricial birds continue to develop adaptive immune function throughout the nestling period, though it is unclear whether nestlings and recent fledglings experience a period of susceptibility to infection before mature adaptive IgY levels are reached.

We present the first study, to our knowledge, to detail the ontogeny of specific adaptive antibody response in an altricial passerine, the zebra finch Taeniopygia guttata, throughout the entire nestling period. Studies of age-related changes in antibody response in zebra finches will contribute knowledge to the fields of comparative immunology and ecological immunology. Additionally, the elaboration of zebra finches as a model is of interest given that their genome has been sequenced and organismal-level studies could provide fundamental knowledge for integrative studies of immune function at a variety of biological levels, including the molecular level. This work aims to address two research questions: i) when are zebra finches capable of mounting a detectable specific secondary antibody response post-hatch? and, ii) does magnitude of secondary adaptive antibody response reach adult levels prior to fledging (21 days post-hatch) in zebra finches? We vaccinated zebra finch nestlings and adults with keyhole limpet hemocyanin (KLH) and detected adaptive KLH-specific antibody response using ELISA. KLH is a model antigen in immunology studies and is a T-cell dependent antigen, providing insight into the integrated ontogeny of both B- and T-cell mediated responses in zebra finches [12], [18]. We used guidelines from previous studies of immunological development in altricial birds [12], [17] as well as a preliminary study using adult male zebra finches to ensure effective stimulation of the immune system of developing zebra finch nestlings and detection of an antibody response, if present.

Methods

Results

Baseline absorbance values from pre-vaccination samples, which likely measure cross-reactivity of ELISA reagents with non-KLH specific antibodies in samples, significantly differed among age groups (F_3,44∶ 6.5, p<0.001). Adults (0.179±0.026) had significantly higher baseline absorbance values than all of the nestling groups. Nestlings at 7d (0.106±0.007), 14d (0.111±0.016), and 21d (0.096±0.006) post-hatch did not significantly differ from each other in baseline absorbance values. To correct for differences among age groups, baseline absorbance values were subtracted from post-priming vaccination and post-boost antibody response values for each bird (or sibling bird in the case of the 7d group).

Repeated measures analysis of corrected KLH antibody response revealed a significant interaction of age and sampling time (Chi-square: 53.94, d.f. 3, p<0.001) (Figure 1). Comparisons within an age group revealed no significant increase in KLH-specific antibody levels between vaccination and boost in 7d birds (p = 0.992), yet highly significant increases between vaccination and boost were observed in birds vaccinated at 14d (p = 0.005) and 21d (p<0.001) and as adults (p<0.001).

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Figure 1. KLH-specific antibody response in vaccinated zebra finch nestlings and adults.

Data are means ± s.e.m. (n = 13 for 7d and 14d groups, n = 14 for 21d and adult groups). Post-priming vaccination (unfilled bars) and post-boost (filled bars) absorbance values were corrected for absorbance values of baseline blood samples on KLH-specific ELISA. Post-vaccination values among age groups that share the same lowercase letter and post-boost values among age groups that share the same uppercase letter are not significantly different as determined by Tukey’s post hoc tests. Significant differences between post-vaccination and post-boost values within an age group (as determined by Tukey’s post hoc tests) are denoted by asterisks.

https://doi.org/10.1371/journal.pone.0047294.g001

There was no significant difference among age groups in corrected KLH antibody levels in response to priming vaccination (Figure 1), yet there were significant differences in corrected KLH antibody levels post-boost, with responses at 7d<14d<21d< adults (Figure 1). Post-boost antibody responses in all nestling age groups were significantly lower than in adults, indicating that mature adult secondary antibody response level was not achieved in zebra finches prior to fledging (Figure 1).

Hematocrit (Chi-square <0.001, d.f. 1, p>0.99) and body mass (Chi-square: <0.001, d.f. 1, p: 0.994) were not significant factors in predicting KLH-specific antibody response. Parental breeding pair was a significant random effect for corrected antibody response (Chi-square: 5.42, d.f. 1, p: 0.020). Vaccination did not significantly impact body mass or hematocrit in growing birds, as birds in different vaccination groups showed no significant differences in body mass or hematocrit measures at a given blood sampling age (data not shown).

Discussion

We present the first study, to our knowledge, to detail the age-related changes in adaptive antibody response in an altricial passerine. Adaptive antibody response was not detectable in birds first injected at 7 days post-hatch, as there was a lack of significant increase in KLH-specific IgY between priming injection and boost. Significantly elevated levels of KLH IgY were detected post-boost in 14d, 21d and adult age groups, indicating capacity for adaptive antibody response. Lack of significant increase in 7d birds under our sampling regimen may be a result of an incompletely developed cell system, resulting in a more prolonged process of Ig class-switching in younger birds. Turkey poults injected with Brucella abortus antigen on the day of hatch reached peak antibody response at 15 days post-injection (dpi), whereas the specific antibody response of birds injected at 3 weeks post-hatch peaked sooner at 7dpi [15], indicating that the number and the functional competence of the B-cells likely increased with age. Detailed study of lymphocyte subpopulations and of the kinetics of antibody response over a range of times post-injection would shed light on the time course of Ig class switching and of the response of specific memory B-cell clones in altricial birds at different ages post-hatch.

In agreement with a previous study in American kestrels, we found that magnitude of adaptive antibody response gradually increases with age, yet is significantly lower than mature adult levels throughout the nestling period. We found that adaptive antibody response in zebra finches did not reach adult levels prior to fledging (21 days post-hatch in zebra finches), when free-living birds leave the nest and experience a more diverse antigenic environment.

Given that nestling birds have limited adaptive immune defenses, they may rely on other immune defenses for resistance. The lack of mature functional antibody response may be partly compensated by maternal provisioning of IgY via the egg yolk, which is absorbed into nestling circulation during development. However, studies of altricial passerine nestlings have shown that maternally derived antibodies are catabolized from circulation within two weeks post-hatch [7]–[9], which, according to our study, overlaps with the time that nestlings have immature endogenous humoral immune response capacity. Lack of mature adaptive immune function in growing nestlings may be compensated by endogenous innate immune defenses because their development does not require generation of diverse receptor specificities or memory cell lines [21], [22]. However, a recent study of tree swallows throughout the nestling period demonstrated that the bactericidal ability of whole blood was at sub-adult level at fledging age [23]. Levels of other innate immune measures, such as natural antibodies and complement, have also been shown to be at immature levels near fledging age in altricial passerines [24], [25]. Alternatively, nestlings may rely more on tolerance of infection while immune system components develop. Given that birds early post-hatch are overwhelmed with large quantities of novel foreign antigen and have high energetic demands of growth and development, it may be energetically cheaper to tolerate infection and limit damage rather than invest in immune defenses [26]. Given these previous studies of altricial passerines, this study provides another instance of immunological immaturity and is consistent with the hypothesis that passerine birds are particularly susceptible throughout the nestling period and immediately after fledging. More integrated studies of age-related changes in both passive and active components of immune resistance as well as tolerance will continue to shed light on the development of effective immune defense in growing altricial birds.

Knowledge of the ontogeny of adaptive immune defense in altricial passerines could have important implications for disease ecology. Passerines play an important role in the spread of zoonotic diseases in the wild, and nestling passerines in particular may be important hosts for vector-borne diseases given their undeveloped defenses and confinement to nests in close proximity to others, making them vulnerable to virus-carrying insects [27], [28]. Because detection of active viral infections can be difficult [28], wild birds are often caught and screened for virus-specific antibodies to provide evidence of a past infection. Knowledge of the ontogeny of antibody response can contribute to understanding of patterns of natural viremia in free-living nestlings and to effectively evaluate nestling exposure to zoonotic diseases.

A number of studies have examined the link between body mass and immune response in young birds, to examine proposed trade-offs associated with growth and immune processes competing for shared resources [29]. Adaptive humoral defenses are proposed to incur low costs of use because they are associated with anti-inflammatory cytokines [1], [30]. However, development of the adaptive humoral components may be relatively costly, given that generation and rapid proliferation of B-cell lineages with diverse, functional receptor specificities is confined to the energetically demanding developmental period of most vertebrates [30]. Reduced body mass growth was observed in young antigen-challenged precocial quail [31] yet vaccination did not interfere with growth in challenged altricial American kestrels [12] or the zebra finches in our study. Because the developing birds in our study were provided with ad libitum food, it is difficult to evaluate the relative costs of immunity. A study of skin-swelling response to phytohemagglutinin (PHA) in captive sand martins (Riparia riparia) demonstrated that the magnitude of PHA response was positively correlated with body mass growth when animals were fed ad libitum and negatively correlated with body mass growth when food was restricted 40% for three days [32], highlighting the importance of considering energy intake when evaluating trade-offs in growth and immune response. Future studies of the ontogeny of immune function in birds should examine the relationship between growth and antibody response in the context of a variety of ecologically relevant feeding scenarios to examine the biological costs of developing and mounting specific antibody responses and the sustained impact of environmental perturbations on immune defense and fitness in free-living animals.

Lastly, we found that parental breeding pair was a significant predictor of antibody response, perhaps indicating a genetic component to the magnitude of antibody response. Genetic-based constraints have been posited to influence the strength of immune response [33] and likely contribute to variability in antibody response to diverse antigens in growing birds [14], [16], [33]. Future experiments should include use of cross-fostering designs or examination of genes involved in recognition (including highly polymorphic MHC loci) to more deliberately examine the contribution of genetic constraints to variability in immune response during development in altricial birds. Additionally, comparative studies of diverse antigens (e.g. live infection versus inert antigens, T-cell dependent versus independent antigens) could contribute knowledge to variability in immune ontogeny in growing birds.

Acknowledgments

We thank undergraduate research assistants and the UW Animal Sciences Care Staff for assistance with bird husbandry. Thanks also to Erik Hofmeister and Karasov lab members for their comments on the experimental design, and to anonymous reviewers for comments on earlier versions of this manuscript.