lymphocyte ratios predict the magnitude of humoral immune response to a novel antigen in great tits (Parus major)

Comparative Biochemistry and Physiology, Part A 161 (2012) 422–428

Contents lists available at SciVerse ScienceDirect

Comparative Biochemistry and Physiology, Part A journal homepage: www.elsevier.com/locate/cbpa

Heterophil/lymphocyte ratios predict the magnitude of humoral immune response to a novel antigen in great tits (Parus major) Indrikis Krams a, b, c, Jolanta Vrublevska b, Dina Cirule b, d, Inese Kivleniece b, Tatjana Krama b, Markus J. Rantala c, Elin Sild a, Peeter Hõrak a,⁎ a

Institute of Ecology and Earth Sciences, University of Tartu, 51014 Tartu, Estonia Institute of Systematic Biology, University of Daugavpils, LV-5401 Daugavpils, Latvia Section of Ecology, Department of Biology, University of Turku, FIN-20024 Turku, Finland d Institute of Food Safety, Animal Health and Environment BIOR, LV-1076, Riga, Latvia b c

a r t i c l e

i n f o

Article history: Received 4 November 2011 Received in revised form 31 December 2011 Accepted 31 December 2011 Available online 8 January 2012 Keywords: Antibody response Brucella abortus Body mass loss H/L ratio Immune challenge Immunosuppression Parus major Stress

a b s t r a c t Animals display remarkable individual variation in their capacity to mount immune responses against novel antigens. According to the life-history theory, this variation is caused by the costs of immune responses to the hosts. We studied one of such potential costs, depletion of somatic resources in wintering wild-caught captive passerines, the great tits (Parus major) by immune challenging the birds with a novel antigen, killed Brucella abortus (BA) suspension. We found that despite mild temperature conditions in captivity and ad libitum availability of food, immune challenge depleted somatic resources (as indicated by a body mass loss) and elevated relative proportion of heterophils to lymphocytes (H/L ratio) in the peripheral blood of birds. However, body mass loss did not covary with an increase in H/L ratios between two sampling events, which indicates that these two markers of health state describe different aspects of individual physiological condition. Antibody titres were not associated with the extent of body mass loss during the development of immune response, which shows that the somatic cost of immune response was not proportional to the amount of antibody produced. Birds with high pre-immunisation H/L ratios mounted weaker antibody response, which is indicative of stress-induced suppression of humoral immune response and is consistent with the concept of an antagonistic cross-regulation between different components of the immune system. The latter finding suggests a novel diagnostic value of H/L ratios, which reinforces the utility of this simple haematological index for prediction of the outcomes of complicated immune processes. © 2012 Elsevier Inc. All rights reserved.

1. Introduction Parasites and pathogens pose an omnipresent threat to all living organisms. To handle these challenges, hosts rely on various defences, the most complex and powerful of these being the immune system. Interacting broadly with other organismal functions and possessing sophisticated, potentially self-injuring and energy-demanding mechanisms for destruction or elimination of pathogens, the immune system is likely to be costly to develop, maintain, and use. According to the life-history theory, such costs inevitably result in physiological trade-offs in allocation of resources between immune function and other components of fitness (e.g., Sheldon and Verhulst, 1996; Lochmiller and Deerenberg, 2000; Norris and Evans, 2000). Identifying the nature of and currencies involved in the costs of immunity is important for understanding the ecological and evolutionary causes and consequences of variation in immune responsiveness and parasite resistance (Schulenburg et al., 2009; Martin et al., 2011). Two

⁎ Corresponding author. Tel.: + 372 7375075; fax: + 372 7375830. E-mail address: [email protected] (P. Hõrak). 1095-6433/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpa.2011.12.018

main types of the costs accompanying immune system activation are increased energy expenditure (e.g., Lochmiller and Deerenberg, 2000) and immunopathology resulting from the collateral damage to the host inflicted by immune responses (e.g., Råberg et al., 1998). Current study addresses the questions about the possible physiological costs of mounting an immune response in terms of depletion of somatic resources. Traditional approach for assessment of the costs and strength of immune responses involves challenging the experimental animals with a foreign antigen and measurement of the induced responses (reviewed by Boughton et al., 2011; Demas et al., 2011b). Using artificial antigens enables distinguishing the physiological effects of immune challenge from the pathological effects of parasites per se. Most common antigens that enable easy quantification of the magnitude of immune response involve xenogenous erythrocytes (e.g., sheep red blood cells, SRBC), toxic compounds of bacteria (e.g., lipopolysaccharide, LPS; tetanus and diphtheria toxins), plants (e.g., phytohaemagglutinin, PHA; pokeweed extract) and animals (keyhole limpet hemocyanin, KLH). Such a diversity of antigen use in an immunoecological research poses a problem of interpretation and generalisation of the results obtained in different models (Norris and Evans, 2000). For instance,

I. Krams et al. / Comparative Biochemistry and Physiology, Part A 161 (2012) 422–428

without knowing the relative pyrogenicity of different antigens, it would be difficult to decide whether the results of similar experiments using different antigens differ primarily because of the properties of the antigens or other experimental conditions (e.g., Ots et al., 2001). The first step in sorting out these issues would be comparison of physiological impact of different antigens. Of the physiological parameters affected by immune challenge, the most easily measurable is the body mass change. Consequently, one of the aims of this study is to measure the effect of immune challenge with a Brucella abortus (BA) antigen on body mass dynamics of wild-caught wintering great tits (Parus major) and compare this with a published record on other avian models. BA belongs to the traditional research tools of poultry immunologists (e.g., Leshchinsky and Klasing, 2001). To our knowledge, only three laboratories have used BA antigen in immunoecological research of wild bird species (Birkhead et al., 1998; Amat et al., 2007; Sild and Hõrak, 2010). However, usage of this antigen possesses several advantages such as the ease of measurement of antibody titres by agglutination test (Munns and Lamont, 1991), minimal effort required for preparation of injection solution (e.g., compared to triple washing of SRBC shortly before injection), and availability of standard antigen stocks (e.g., compared to possible donor-related variation in immunogenicity of erythrocytes or bacterial LPS). The second aim of this study was to describe the associations between the magnitude of antibody response to BA, body mass and heterophil/ lymphocyte (H/L) ratio. The question about energetic costs of antibody production is not fully understood in the immunoecological literature. On the one hand, the quantitative need for nutrients for the proliferation of leucocytes and production of antibodies has been estimated very small (Klasing, 1998). On the other hand experimental elevation of work load (reviewed by Hasselquist et al., 2001; Knowles et al., 2008), or energy expenditure (Svensson et al., 1998) has suppressed antibody response in some avian studies, although not always (Hasselquist et al., 2007). However, there is very little evidence that individuals in superior condition are capable of mounting stronger antibody responses, as most studies have found no association between individual body condition or mass and antibody production (reviewed by Møller and Petrie, 2002) (but see Saks et al., 2006; Bourgeon et al., 2010). Findings of those studies (majority of which have measured antibody responses to SRBC or tetanus–diphtheria vaccine) challenge the general concept that immune responsiveness is condition-dependent (Gustafsson et al., 1994; Møller et al., 1998; Råberg et al., 1998; Demas et al., 2011a). We were thus interested in whether the antibody production against BA appears similarly condition-independent as antibody responses to other standard antigens. As an additional index of individual physiological condition, we measured the H/L ratios before and after the antigen injection. Heterophils and lymphocytes comprise the majority of circulating immune cells in birds and H/L ratios are specifically sensitive to either natural stressors or administration of stress hormones, so that relatively high heterophil counts in relation to lymphocytes reliably indicate high glucocorticoid levels (reviewed by Ots et al., 1998; Davis et al., 2008). Assessment of stress on the basis of H/L ratios has several advantages over measurement of hormone levels (Davis et al., 2008) and high H/L ratios have been associated with increased mortality in the field (reviewed by Sepp et al., 2010). According to the concept of glucocorticoid-induced immunosuppression in chronic stress (Maier et al., 1994; Sapolsky et al., 2000; Dhabhar, 2009), we predicted that less stressed individuals, i.e., the birds with initially low H/L ratios would mount stronger antibody responses to BA than the birds with high H/L ratios. This prediction relies on the assumption that H/L ratios at capture reflect the extent of chronic stress experienced previously (see Krams et al., 2011). 2. Materials and methods The study was conducted in the city of Daugavpils (55°52′N, 27°12′E), south-eastern Latvia. The data were collected during winter of 2009/2010 when ambient temperatures often dropped to −30 °C during the night.

423

Yearling male great tits were captured with mist nets 1 h before sunset at permanent feeders baited with sunflower seeds on 17.12.09. (22 individuals) and 04.01.10. (17 individuals). Minimum night temperatures on these dates were −27 °C and −12 °C and minimum daily temperatures were −6 °C and −2 °C, respectively. Birds were transported to the aviary and housed individually into cages (20×30×40 cm) with sand bedding. They were supplied ad libitum with sunflower seeds and tap water; each bird also received three mealworms per day. Birds were held on the natural day-length cycle on artificial lighting. Average temperature in the aviary during the experiment was 8.0±1.9 °C (SD). Birds caught on 17th of December, were randomly assigned to immune challenge or control group (12 vs 10 individuals, respectively) at arrival to the aviary. All the birds caught on 4th of January were immune challenged. Before immunisation treatment, the birds were weighed with a precision of 0.01 g and blood sampled (from tarsal vein) for making blood smears and determination of antibodies. Serum for determination of antibodies was separated by centrifugation from 100 μL of blood, collected into Microvette tubes and stored at −75 °C until analysed. After blood sampling the birds in the immune-challenged group (altogether 29 individuals) received an injection of 50 μL of B. abortus (BA) vaccine into the pectoral muscle (B. abortus vaccine strain RB-51, product code 28710, Boehringer Ingelheim Pharmaceuticals, USA). Birds in the control group (10 individuals) were injected with the same amount of isotonic saline. In the afternoon of day 6 (day 0=day of capture) the birds were weighed and blood sampled again and released into their natural habitat in the following morning. Antibody response to BA was quantified according to the method of Amat et al. (2007) and Munns and Lamont (1991) with minor modifications. Briefly, 45 μL of serum was added to 45 μL of phosphate-based saline (PBS) (Sigma P-4417) in the first well of a 96 well microtitre plate and serially doubled dilutions in PBS (1:2, 1:4, 1:8 etc.) were then carried out in the consecutive wells. Then 45 μL of BA serum Agglutination Test Antigen was added to each well. After incubation of plates for 24 h at room temperature, agglutination was visually scored. Antibody titre was scored as the number of wells in a dilution row that contained a sufficient amount of antibodies to agglutinate B. abortus. BA positive (RAB1003, Veterinary Laboratories Agency, Weybridge, UK) and negative (RAB0701, Veterinary Laboratories Agency, Weybridge, UK) serums were used as controls. None of the nonimmunised birds produced a detectable amount of antibodies against BA. Blood smears for differential leukocyte counts were prepared using the standard two-slide wedge procedure. The samples were air dried, fixed in methanol and stained with Wright-Giemsa Quick stain. Smears were examined to obtain counts of lymphocytes and granulocytes per 100 leukocytes. Obtained cell counts were used for calculation of the relative proportion of heterophils to lymphocytes (H/L ratios). Effects of immune challenge on changes in H/L ratio and body mass between the first and second blood sampling were tested in ANCOVAs adjusting for the initial trait values (Table 1). Assumptions for parametric models (normality of residuals, homogeneity of variances)

Table 1 Effects of BA and saline injection on body mass and H/L changes of great tits over 6 days in an ANCOVA adjusting for initial trait values. Interaction terms between immune challenge and initial trait values were not significant (P = 0.2). η2 is coefficient of partial determination, describing the proportion of total variation attributable to the predictor variable, partialling out other factors from the total non error variation. Dependent variable

Predictors

df

F

η2

P

Body mass change

Initial body mass Immune challenge ln (initial H/L) Immune challenge Initial H/L H/L increase (H/L increase)2

1,36 1,36 1,36 1,36 1,25 1,25 1,25

49.5 5.3 21.4 10.2 12.7 15.9 16.7

0.58 0.13 0.37 0.22 0.33 0.39 0.40

b 0.00001 b 0.00001 b 0.00001 0.003 0.002 0.0005 0.0003

ln (H/L change) Anti-BA titre

424

I. Krams et al. / Comparative Biochemistry and Physiology, Part A 161 (2012) 422–428

Initial body mass or its change was not associated with antibody production against BA (Fig. 2, A and B). Birds with initially low H/L ratios produced more anti-BA antibodies than the birds with initially high H/L values (Fig. 2C). Relationship between BA titre and increase in H/L was concave: BA titres initially increased with increasing H/L but then dropped at extremely high values of H/L increase (Fig. 2D, Table 1). This decline was caused by three individuals, whose H/L ratio increased above 4 units. However, we did not find that these individuals had differed from the others with respect to any other measured parameters, so we had no reason to exclude them from the analysis. The change in body mass was not associated with the change in H/L ratio, neither in the whole sample (rs = 0.04, P = 0.82, N = 39) nor among the immune challenged birds separately (rs = − 0.03, P = 0.90, N = 29).

were met for all the models. For the H/L change, this required lntransformation. Associations between BA antibody titres and various predictor variables were assessed on the basis of Spearman rank correlation analysis except for the H/L change. In the latter case, quadratic polynomial was fitted (Table 1, Fig. 2D); again assumptions for parametric models being met. Day of capture was not significant in any model, and so was dropped from the final analyses. α levels of 0.05 are used as the criterion of statistical significance. Averages are reported with±SD.

3. Results Initial body masses did not differ between BA-injected and salineinjected birds (19.1± 1.0 g, N = 29 vs 19.4 ± 1.0 g N = 10; t = −0.78; P = 0.44; Fig. 1A). During the six-day period between two samplings BA-injected birds lost on average 2.2± 1.4 g while saline-injected birds lost on average 1.8 ± 1.2 g of body mass. Thus, BA-injected birds lost on average 33% more weight than un-injected birds (2.28 ± 0.16 g vs 1.53± 0.28 g; least square means from the models accounting for initial body mass). This difference was highly significant (Table 1; Fig. 1A). Initial H/L ratios of BA-injected and saline-injected birds were similar (1.17 ± 0.84, N = 29 vs 1.95 ± 1.62 N = 10; z = − 1.48; P = 0.14; U-test; Fig. 1). During the period between two samplings, H/L ratios of BA-injected birds increased on average by 55% while H/L ratios of saline-injected birds decreased on average by 28%. This difference was highly significant (Table 1; Fig. 1B).

A Body mass g

Immune challenge with killed B. abortus significantly reduced the body mass of wild-caught wintering great tits over the 6-day period. This result is comparable to the most of studies reviewed in the Table 2. Thus BA appears to induce similar somatic costs in wild birds as the traditionally used antigens. Most likely explanation for the immunisation-induced mass loss is the anorexia accompanying acute phase response to the antigen (Lochmiller and Deerenberg, 2000; Owen-Ashley and Wingfield, 2007). However, the occurrence of catabolic effects induced by the acute inflammatory processes

22

22

Saline

BA

21

21

20

20

19

19

18

18

17

17

16

16

159 8

H/L ratio

4. Discussion

B

Day 0

Day 6

159

BA

Day 0

8

7

7

6

6

5

5

4

4

3

3

2

2

1

1

DaySaline 6

0

0

Day 0

Day 6

Day 0

Day 6

Fig. 1. Individual changes in body mass and H/L ratio in great tits injected with BA (N = 29) or saline (N = 10) over 6-day period. Whiskers are means ± SE. See Table 1 for P-values.

I. Krams et al. / Comparative Biochemistry and Physiology, Part A 161 (2012) 422–428

10

A A9 BA antibody titers

9

BA antibody titers

8 7 6 5 4

BB

8 7 6 5 4 3

3 2

Irs=-0.12, p=0.5 17

18

Irs=0.17, p=0.4

19

20

-5

-4

10

10

9

C C 9

8 7 6 5 4

Irs=-0.46, p=0.012 1.5

-2

-1

0

1

2.5

3.5

Initial H/L

D D

8 7 6 5 4 3

3

0.5

-3

Mass change (g)

BA antibody titers

BA antibody titers

Initial mass (g)

2

425

2

Irs=0.29, p=0.12 0

1

2

3

4

5

6

7

H/L change

Fig. 2. Covariation between pre-immunisation body mass and H/L ratio and their corresponding changes with BA antibody titres. N = 29. The line in D is a quadratic polynomial (see Table 1).

(Klasing and Austic, 1984) cannot be excluded either (although these are not always observed (Coon et al., 2011)). Furthermore, immune challenges do not always lead to mass loss (Table 2), and may even increase the food intake (Barbosa and Moreno, 2004; Coon et al., 2011). Thus, firm conclusions about the effects of immune challenge on body mass changes are difficult to draw at present because several factors such as species, antigens and life-history stages are largely confounded. Similarly to the majority of previous studies in wild birds (see introduction), we did not find that initially heavier birds would mount stronger antibody responses to BA. All these findings can have several mutually non-exclusive explanations. First, body mass may not appear a suitable proxy for individual physiological condition or wellbeing in the case of wintering great tits. However, the evidence from our study population suggests the opposite, since low body masses and fat scores are pertinent to subordinate individuals under harsh climate conditions (Krams, 2000; Krams et al., 2010). Second, the production of antibodies per se may be energetically or nutritionally cheap, so that producing a high amount of immunoglobulins does not constitute a major challenge for an individual short on bodily reserves. For instance, zebra finches (Taeniopygia guttata) subjected to food restriction and enforced physical exercise produced more antibodies in response to SRBC (but not to BA) than control birds, despite higher H/L ratios indicating that experimental birds were more stressed (Birkhead et al., 1998). An opposite example is presented by the study of freeliving wintering great tits (Ots et al., 2001) and captive collared doves

(Streptopelia decaocto; Eraud et al., 2005) where the birds that mounted stronger antibody responses to SRBC also lost more weight. Hence, under some specific conditions, somatic costs of humoral immune response may be proportional to the amount of antibody produced. Such pattern did not emerge in the current study despite substantially higher average mass loss in captive conditions (Fig. 2B) as compared to the study of Ots et al. (2001) in free-living great tits. This discrepancy seems to be at odds with the general logic of the life-history theory that correlational trade-offs should be at first place revealed in situations where between-individual variation in resource acquisition is relatively small (Van Noordwijk and de Jong, 1986), as one might expect in the case of captive conditions. Further, the relationships between individual body condition and the magnitude of antibody response may also depend on other factors which remain un-registered in most of studies. For instance, Saks et al. (2006) found that anti-SRBC antibody titres correlated positively with initial body mass only among the birds that were not susceptible to an experimental infection with heterologous coccidian strains. Immune challenge with BA significantly increased relative proportion of heterophils to lymphocytes. The increase in the number of circulating heterophils is generally considered a symptom of inflammatory and stress responses (e.g., Gross and Siegel, 1983; Campbell and Ellis, 2007). Its main mediators are pro-inflammatory cytokines released by activated macrophages; these cytokines mediate changes in glucocorticoids, acutephase proteins, and recruitment of monocytes and heterophils from the bone marrow (Leshchinsky and Klasing, 2001). Heterophils play an important role in innate immune response. These phagocytes are the first

426

I. Krams et al. / Comparative Biochemistry and Physiology, Part A 161 (2012) 422–428

Table 2 Effects of immune challenge with novel antigens on body mass change in wild birds. Species

Somateria mollissima Streptopelia decaocto Parus major Carduelis chloris

Carduelis chloris

Carduelis chloris Growing Coturnix coturnix japonica

Calidris canutus Philomachus pugnax Parus caeruleus

Hirundo rustica nestlings Passer domesticus Streptopelia decaocto nestlings Melospiza melodia morphna

Antigen

SRBC* SRBC SRBC* SRBC autumn SRBC autumn SRBC spring SRBC spring SRBC SRBC* SRBC§ SRBC§ SRBC + BA SRBC + BA SRBC* NDV * MS* DT DT DT DT DT* DT* DT* LPS* LPS* LPS* LPS LPS spring LPS winter*

Time lag in days

Control Initial mass g (n)

%change

Experimental Initial mass g (n)

%change

Reference

8 3 6–10 4 8 0.5 3.5 7 25 † 7 25 † 5 6 10 10 10 14 14 7 14 28 † 35 † 42 † 2 3 6 4 1 1

1908 (30) 193.5 (10) 18.6 (10) 27.4 (16) 27.4 (16) 27.2 (7) 27.2 (7) 29.4 (14) 29.4 (14) 29.6 (14) 29.6 (14) 25.1 (14) 25.1 (14) 102 (8) 102 (8) 102 (8) 116 (9) 97.0 (10) 11.1 (11) 11.1 (11) 11.1 (7) 11.1 (7) 11.1 (7) 20.7 (100) 20.7 (100) 25.1 (10) 42.2 (15) 24.7 (4) 26.1 (6)

− 10.2% − 8.8% − 0.3% − 1.5% − 1.8% − 4.0% − 3.7% − 0.4% + 6.5% + 0.1% + 4.4% 0 + 0.2% + 44.5% + 44.5% + 44.5% + 9.0% + 17.0% − 1.6% −0.2% − 4.5% − 2.7% − 1.8% − 1.5% − 3.6% + 2.4% + 102.1% − 1.6% − 0.7%

1910 (28) 192.5 (10) 18.8 (15) 27.8 (16) 27.8 (16) 27.5 (7) 27.5 (7) 29.2 (14) 29.2 (14) 29.0 (14) 29.0 (14) 24.2 (16) 24.2 (16) 102 (8) 102 (7) 102 (7) 119 (10) 96.6 (10) 11.3 (12) 11.3 (12) 11.3 (7) 11.3 (7) 11.3 (7) 20.7 (102) 20.7 (102) 25.7 (12) 47.1 (16) 24.7 (5) 26.1 (4)

− 11.8% − 9.6% − 2.8% − 2.5% − 3.6% − 3.6% − 5.1% − 0.8% − 0.6% − 0.6% + 1.7% + 0.4% 0 + 32.1% + 19.7% + 25.9% + 7.0% + 16.0% + 0.2% + 1.4% + 6.2% + 6.2% + 5.3% − 3.9% − 4.84% − 1.6% + 95.8% − 2.5% − 5.2%

(Hanssen, 2006) (Eraud et al., 2005) (Ots et al., 2001) (Hõrak et al., 2003)

(Hõrak et al., 2006)

(Amat et al., 2007) (Fair et al., 1999)

(Mendes et al., 2006) (Mendes et al., 2006) (Svensson et al., 1998)

(Romano et al., 2011) (Moreno-Rueda, 2011) (Eraud et al., 2009) (Owen-Ashley and Wingfield, 2006)

NDV killed Newcastle disease virus; MS inactivated Mycoplasma synoviae; DT diphtheria and tetanus vaccine; BA Brucella abortus; § carotenoid-supplemented birds; *significant difference in mass change; † secondary response to antigen after secondary injection

cells to migrate to the site of infection where they engage in phagocytosis and killing of pathogens by producing toxic reactive oxygen species and releasing bactericidal substances and proteolytic enzymes in the process of oxidative burst and degranulation (He et al., 2008). Effect of BA injection on H/L ratio thus indicates induction of strong inflammatory response that lasts at least 6 days. This finding compares favourably with a study on captive greenfinches (Carduelis chloris), showing that BA injection in vivo potentiated the capability of phagocytic cells in whole blood to produce oxidative burst 7 days after immune challenge (Sild and Hõrak, 2010). Altogether these findings confirm that B. abortus is a suitable antigen for assessment of the potential long-term costs of immune activation (see also Ottenweller et al., 1998). Perhaps the most interesting result of the current study is that pre-injection H/L ratios correlated negatively with anti-BA antibody titres (Fig. 2C). To our knowledge, such a correlation is a unique finding, although indirect evidence about stress-induced suppression of antibody production in the studies of poultry (reviewed by Moe et al., 2010) is consistent with the observed pattern. Thus, our study provides evidence against the assertion that ‘the information obtained from one blood smear tells us little about the ability of that individual to mount an immune response’ (Davis et al., 2008). The generality of this finding, however, remains to be tested in different species, antigens and life-history stages. The most parsimonious explanation to our findings is that high H/L ratios are caused by the increased blood corticosterone levels (El Lethey et al., 2003; Müller et al., 2010; Shini et al., 2010a), which also suppress antibody response to BA (Takahashi et al., 1992). Assuming that initially high H/L levels reflect stress, then it is likely that low anti-BA antibody titres of birds with high H/L ratios were caused by down-regulation of pro-inflammatory and up-regulation by anti-inflammatory cytokines by corticosterone (Shini et al., 2010b). Such a situation would resemble a cross-regulation between different

types of immune responses in mammals where a shift from initially predominating Th1-cell-mediated immunity to Th2-response eventually leads to down-regulation of inflammatory response by a positive feedback mechanism (Calcagni and Elenkov, 2006; Shini and Kaiser, 2009). In this context it is notable that in chicken the antibody response to BA antigen is mediated by the Th1 (pro-inflammatory) cytokine Interferon-γ (Zhou et al., 2001; see also Golding et al., 2001; Oliveira et al., 2008). Administration of corticosterone to chickens, in turn, significantly down-regulated Interferon-γ mRNA expression 24 h and 1 week after treatment (Shini and Kaiser, 2009). We might thus speculate that similar molecular events could have been responsible for the negative correlation between H/L ratio and antibody response in the current study. The ultimate function of such an interference between the stress system and immune and inflammatory response is probably protection of an organism from systemic ‘overshooting’ with Th1proinflammatory cytokines and avoidance of accompanying immunopathology (Calcagni and Elenkov, 2006). Notably, in a study of wildcaught greenfinches, individuals that tolerated the captivity better (i.e., those that resorted to a lesser extent to flapping flights against cage walls) mounted stronger antibody response to BA than less captivity-tolerant birds (Sild et al., 2011). Similarly to the current study, these results point to the connection between immune responsiveness to BA and stress tolerance. The non-linear relationship between an increase in H/L ratio and antibody titres (Fig. 2D) is more difficult to interpret. It might be possible that three birds with the highest increase in H/L ratios that showed lower antibody response than those with intermediate increase in H/L perceived particularly severe psychological stress or suffered a relapse of some opportunistic infection. For instance, bacterial LPS can increase H/L ratios similarly to corticosterone, but probably through different mechanisms (Shini et al., 2008). Notably, the increase in H/L ratio did not correlate with body mass loss in captivity, suggesting that stress

I. Krams et al. / Comparative Biochemistry and Physiology, Part A 161 (2012) 422–428

level did not increase in parallel with deterioration in body condition. This finding once more indicates that under the current experimental situation, H/L ratio and mass change reflected different aspects of individual physiological condition. This finding differs from that of Ots et al. (2001), who showed that free-living wintering great tits that lost more body mass during the 6–10-day period between blood samplings also had the highest increase in H/L ratios during that period (rs = 0.46, P = 0.025, N = 24). Wild wintering great tits in our study population displayed a negative correlation between H/L ratio and another index of body condition, the breast muscle score; besides subordinate individuals had the highest increase in H/L and the highest decrease in body mass during cold spells (Krams et al., 2011). Experimental fasting of herring gulls (Larus argentatus) for 6 days induced a significant increase in H/L ratio (Totzke et al., 1999). In wild burrowing parrots (Cyanoliseus patagonus) (Plischke et al., 2009) and upland geese (Chloephaga picta leucoptera) (Gladbach et al., 2010) H/L ratios covaried negatively with body condition (mass adjusted for structural size). Current study differs from the previously cited ones by ad libitum access to highly preferred food (sunflower seeds). It may thus appear possible that associations between body mass and H/L ratios in wild birds are caused by the effects of social dominance/subordinance on both stress levels and access to food. In conclusion, this study showed that induction of immune response in great tits with B. abortus killed antigen depletes somatic resources (as indicated by a body mass loss) and elevates relative proportion of heterophils to lymphocytes (H/L ratio) in the peripheral blood. However, body mass loss did not covary with an increase in H/L ratios between two sampling events, which indicates that these two markers of health state reflected different aspects of individual physiological condition. Birds with high pre-immunisation H/L ratios mounted weaker antibody response to BA, which is indicative of stress-induced suppression of humoral immune response and suggests an antagonistic cross-regulation between different components of the immune system. The latter finding suggests a novel diagnostic value of H/L ratios, which reinforces the utility of this simple haematological index for prediction of the outcomes of complicated immune processes (see also Cirule et al., 2012). Therefore, the question whether and under which circumstances H/L ratios predict the magnitude of immune responsiveness in other model systems deserves further attention. Acknowledgements P. Hõrak and E. Sild were financed by Estonian Science Foundation (grant # 7737 to PH), the Estonian Ministry of Education and Science (target-financing project # 0180004s09) and by the European Union through the European Regional Development Fund (Centre of Excellence FIBIR). M. J. Rantala and I. Krams were supported by the Academy of Finland. Latvian Council of Science (grant 09.1186) financed T. Krama, and the European Social Fund within the project ‘Support for the implementation of doctoral studies at Daugavpils University’ Nr.2009/0140/1DP/1.1.2.1.2/09/IPIA/VIAA/ 015 supported Jolanta Vrublevska. We thank Mihails Pupiņš, Sanita Kecko and Valerijs Vahruševs for their help in the field and two anonymous reviewers for their constructive comments on the ms. References Amat, J.A., Aguilera, E., Visser, G.H., 2007. Energetic and developmental costs of mounting an immune response in greenfinches (Carduelis chloris). Ecol. Res. 22, 282–287. Barbosa, A., Moreno, E., 2004. Cell-mediated immune response affects food intake but not body mass: an experiment with wintering great tits. Ecoscience 11, 305–309. Birkhead, T.R., Fletcher, F., Pellatt, J., 1998. Sexual selection in the zebra finch Taeniopygia guttata: condition, sex traits and immune capacity. Behav. Ecol. Sociobiol. 44, 179–191. Boughton, R.K., Joop, G., Armitage, S.A.O., 2011. Outdoor immunology: methodological considerations for ecologists. Funct. Ecol. 25, 81–100.

427

Bourgeon, S., Kauffmann, M., Geiger, S., Raclot, T., Robin, J.-P., 2010. Relationships between metabolic status, corticosterone secretion and maintenance of innate and adaptive humoral immunities in fasted re-fed mallards. J. Exp. Biol. 213, 3810–3818. Calcagni, E., Elenkov, I., 2006. Stress system activity, innate and T helper cytokines, and susceptibility to immune-related diseases. Ann. N.Y. Acad. Sci. 1069, 62–76. Campbell, T.W., Ellis, C., 2007. Avian and exotic animal hematology and cytology. Blackwell, Oxford. Cirule, D., Krama, T., Vrublevska, J., Rantala, M., Krams, I., 2012. A rapid effect of handling on counts of white blood cells in a wintering passerine bird: a more practical measure of stress? J. Ornithol. 153, 161–166. Coon, C.A.C., Warne, R.W., Martin, L.B., 2011. Acute-phase responses vary with pathogen identity in house sparrows (Passer domesticus). Am. J. Physiol. 300, R1418–R1425. Davis, A.K., Maney, A.K., Maerz, J.C., 2008. The use of leukocyte profiles to measure stress in vertebrates: a review for ecologists. Funct. Ecol. 760–772. Demas, G.E., Adamo, S.A., French, S.S., 2011a. Neuroendocrine-immune crosstalk in vertebrates and invertebrates: implications for host defence. Funct. Ecol. 25, 29–39. Demas, G.E., Zysling, D.A., Beechler, B.R., Muehlenbein, M.P., French, S.S., 2011b. Beyond phytohaemagglutinin: assessing vertebrate immune function across ecological contexts. J. Anim. Ecol. 80, 710–730. Dhabhar, F.S., 2009. Enhancing versus suppressive effects of stress on immune function: implications for immunoprotection and immunopathology. Neuroimmunomodulation 16, 300–317. El Lethey, H., Huber-Eicher, B., Jungi, T.W., 2003. Exploration of stress-induced immunosuppression in chickens reveals both stress-resistant and stress-susceptible antigen responses. Vet. Immunol. Immunopathol. 95, 91–101. Eraud, C., Duriez, O., Chastel, O., Faivre, B., 2005. The energetic cost of humoral immunity in the collared dove, Streptopelia decaocto: is the magnitude sufficient to force energybased trade-offs? Funct. Ecol. 19, 110–118. Eraud, C., Jacquet, A., Faivre, B., 2009. Survival cost of an early immune soliciting in nature. Evolution 63, 1036–1043. Fair, J.M., Hansen, E.S., Ricklefs, R.E., 1999. Growth, developmental stability and immune response in juvenile Japanese quails (Coturnix coturnix japonica). Proc. R. Soc. B Biol. Sci. 266, 1735–1742. Gladbach, A., Gladbach, D.J., Quillfeldt, P., 2010. Variations in leucocyte profiles and plasma biochemistry are related to different aspects of parental investment in male and female upland geese Chloephaga picta leucoptera. Comp. Biochem. Physiol. A 156, 269–277. Golding, B., Scott, D.E., Scharf, O., Huang, L.Y., Zaitseva, M., Lapham, C., Eller, N., Golding, H., 2001. Immunity and protection against Brucella abortus. Microb. Infect. 3, 43–48. Gross, W.B., Siegel, H.S., 1983. Evaluation of the heterophil/lymphocyte ratio as a measure of stress in chickens. Avian Dis. 27, 972–979. Gustafsson, L., Nordling, D., Andersson, M.S., Sheldon, B.C., Qvarnström, A., 1994. Infectious diseases, reproductive effort and the cost of reproduction in birds. Philos. Trans. R. Soc. London B Biol. Sci. 346, 323–331. Hanssen, S.A., 2006. Costs of an immune challenge and terminal investment in a long-lived bird. Ecology 87, 2440–2446. Hasselquist, D., Wasson, M.F., Winkler, D.W., 2001. Humoral immunocompetence correlates with date of egg-laying and reflects work load in female tree swallows. Behav. Ecol. 12, 93–97. Hasselquist, D., Lindström, A., Jenni-Eiermann, S., Koolhaas, A., Piersma, T., 2007. Long flights do not influence immune responses of a long-distance migrant bird: a wind-tunnel experiment. J. Exp. Biol. 210, 1123–1131. He, H.Q., Genovese, K.J., Swaggerty, C.L., Nisbet, D.J., Kogut, M.H., 2008. Differential induction of nitric oxide, degranulation, and oxidative burst activities in response to microbial agonist stimulations in monocytes and heterophils from young commercial turkeys. Vet. Immunol. Immunopathol. 123, 177–185. Hõrak, P., Saks, L., Ots, I., Kullissaar, T., Kollist, H., Zilmer, M., 2003. Physiological effects of immune challenge in captive greenfinches (Carduelis chloris). Can. J. Zool. 81, 371–379. Hõrak, P., Zilmer, M., Saks, L., Ots, I., Karu, U., Zilmer, K., 2006. Antioxidant protection, carotenoids and the costs of immune challenge in greenfinches. J. Exp. Biol. 209, 4329–4338. Klasing, K.C., 1998. Nutritional modulation of resistance to infectious diseases. Poult. Sci. 77, 1119–1125. Klasing, K.C., Austic, R.E., 1984. Changes in plasma, tissue and urinary nitrogen metabolites due to inflammatory challenge. Proc. Soc. Exp. Biol. Med. 176, 276–284. Knowles, S.C.L., Nakagawa, S., Sheldon, B.C., 2008. Elevated reproductive effort increases blood parasitaemia and decreases immune function in birds: a metaregression approach. Funct. Ecol. 23, 405–415. Krams, I., 2000. Length of feeding day and body weight of great tits in a single-and a two-predator environment. Behav. Ecol. Sociobiol. 48, 147–153. Krams, I., Cirule, D., Suraka, V., Krama, T., Rantala, M.J., Ramey, G., 2010. Fattening strategies of wintering great tits support the optimal body mass hypothesis under conditions of extremely low ambient temperature. Funct. Ecol. 24, 172–177. Krams, I., Cirule, D., Krama, T., Vrublevska, J., 2011. Extremely low ambient temperature affects haematological parameters and body condition in wintering great tits (Parus major). J. Ornithol. 152, 889–895. Leshchinsky, T.V., Klasing, K.C., 2001. Relationship between the level of dietary vitamin E and the immune response of broiler chickens. Poult. Sci. 80, 1590–1599. Lochmiller, R.L., Deerenberg, C., 2000. Trade-offs in evolutionary immunology: just what is the cost of immunity? Oikos 88, 87–98. Maier, S.F., Watkins, L.R., Fleshner, M., 1994. Psychoneuroimmunology: the interface between behavior, brain, and immunity. Am. Psychol. 49, 1004–1017. Martin, L.B., Hawley, D.M., Ardia, D.R., 2011. An introduction to ecological immunology. Funct. Ecol. 25, 1–4.

428

I. Krams et al. / Comparative Biochemistry and Physiology, Part A 161 (2012) 422–428

Mendes, L., Piersma, T., Hasselquist, D., 2006. Two estimates of the metabolic costs of antibody production in migratory shorebirds: low costs, internal reallocation, or both? J. Ornithol. 147, 274–280. Moe, R.O., Guemene, D., Bakken, M., Larsen, H.J.S., Shini, S., Lervik, S., Skjerve, E., Michel, V., Tauson, R., 2010. Effects of housing conditions during the rearing and laying period on adrenal reactivity, immune response and heterophil to lymphocyte (H/L) ratios in laying hens. Animal 4, 1709–1715. Møller, A.P., Petrie, M., 2002. Condition dependence, multiple sexual signals, and immunocompetence in peacocks. Behav. Ecol. 13, 248–253. Møller, A.P., Christe, P., Erritzøe, J., Mavarez, J., 1998. Condition, disease and immune defence. Oikos 83, 301–306. Moreno-Rueda, G., 2011. Trade-off between immune response and body mass in wintering house sparrows (Passer domesticus). Ecol. Res. 26, 943–947. Müller, C., Jenni-Eiermann, S., Jenni, L., 2010. Heterophils/lymphocytes-ratio and circulating corticosterone do not indicate the same stress imposed on Eurasian kestrel nestlings. Funct. Ecol. 25, 566–576. Munns, P.L., Lamont, S.J., 1991. Effects of age and immunisation interval on the anamnestic response to T-cell-dependent and T-cell-independent antigens in chickens. Poult. Sci. 70, 2371–2374. Norris, K., Evans, M., 2000. Ecological immunology: life history trade-offs and immune defense in birds. Behav. Ecol. 11, 19–26. Oliveira, S.C., de Oliveira, F.S., Macedo, G.C., de Almeida, L.A., Carvalho, N.B., 2008. The role of innate immune receptors in the control of Brucella abortus infection: toll-like receptors and beyond. Microb. Infect. 10, 1005–1009. Ots, I., Murumägi, A., Hõrak, P., 1998. Haematological health state indices of reproducing great tits: methodology and sources of natural variation. Funct. Ecol. 12, 700–707. Ots, I., Kerimov, A.B., Ivankina, E.V., Ilyina, T.A., Hõrak, P., 2001. Immune challenge affects basal metabolic activity in wintering great tits. Proc. R. Soc. London B Biol. Sci. 268, 1175–1181. Ottenweller, J.E., Natelson, B.H., Gause, W.C., Carroll, K.K., Beldowicz, D., Zhou, X.D., LaManca, J.J., 1998. Mouse running activity is lowered by Brucella abortus treatment: a potential model to study chronic fatigue. Physiol. Behav. 63, 795–801. Owen-Ashley, N.T., Wingfield, J.C., 2006. Seasonal modulation of sickness behavior in free-living northwestern song sparrows (Melospiza melodia morphna). J. Exp. Biol. 209, 3062–3070. Owen-Ashley, N., Wingfield, J., 2007. Acute phase responses of passerine birds: characterization and seasonal variation. J. Ornithol. 148 (Suppl. 2), 583–591. Plischke, A., Quillfeldt, P., Lubjuhn, T., Merino, S., Masello, J., 2009. Leucocytes in adult burrowing parrots Cyanoliseus patagonus in the wild: variation between contrasting breeding seasons, gender, and individual condition. J. Ornithol. 151, 347–354. Råberg, L., Grahn, M., Hasselquist, D., Svensson, E., 1998. On the adaptive significance of stress-induced immunosuppression. Proc. R. Soc. London B Biol. Sci. 265, 1637–1641.

Romano, A., Rubolini, D., Caprioli, M., Boncoraglio, G., Ambrosini, R., Saino, N., 2011. Sex-related effects of an immune challenge on growth and begging behavior of barn swallow nestlings. PLoS One 6. Saks, L., Karu, U., Ots, I., Hõrak, P., 2006. Do standard measures of immunocompetence reflect parasite resistance? The case of greenfinch coccidiosis. Funct. Ecol. 20, 75–82. Sapolsky, R.M., Romero, L.M., Munck, A.U., 2000. How do glucocorticoids influence stress responses? Integrating permissive, suppressive, stimulatory, and preparative actions. Endocr. Rev. 21, 55–89. Schulenburg, H., Kurtz, J., Moret, Y., Siva-Jothy, M.T., 2009. Introduction. Ecological immunology. Philos. Trans. R. Soc. B Biol. Sci. 364, 3–14. Sepp, T., Sild, E., Hõrak, P., 2010. Hematological condition indexes in greenfinches: effects of captivity and diurnal variation. Physiol. Biochem. Zool. 83, 276–282. Sheldon, B.C., Verhulst, S., 1996. Ecological immunology: costly parasite defences and trade-offs in evolutionary ecology. Trends Ecol. Evol. 11, 317–321. Shini, S., Kaiser, P., 2009. Effects of stress, mimicked by administration of corticosterone in drinking water, on the expression of chicken cytokine and chemokine genes in lymphocytes. Stress - Int. J. Biol. Stress 12, 388–399. Shini, S., Kaiser, P., Shini, A., Bryden, W.L., 2008. Biological response of chickens (Gallus gallus domesticus) induced by corticosterone and a bacterial endotoxin. Comp. biochem. Physiol. B 149, 324–333. Shini, S., Huff, G.R., Shini, A., Kaiser, P., 2010a. Understanding stress-induced immunosuppression: exploration of cytokine and chemokine gene profiles in chicken peripheral leukocytes. Poult. Sci. 89, 841–851. Shini, S., Shini, A., Kaiser, P., 2010b. Cytokine and chemokine gene expression profiles in heterophils from chickens treated with corticosterone. Stress - Int. J. Biol. Stress 13, 185–194. Sild, E., Hõrak, P., 2010. Assessment of oxidative burst in avian whole blood samples: validation and application of a chemiluminescence method based on Pholasin. Behav. Ecol. Sociobiol. 64, 2065–2076. Sild, E., Sepp, T., Hõrak, P., 2011. Behavioural trait covaries with immune responsiveness in a wild passerine. Brain. Behav. Immun. 25, 1349–1354. Svensson, E., Råberg, L., Koch, C., Hasselquist, D., 1998. Energetic stress, immunosuppression and the costs of an antibody response. Funct. Ecol. 12, 912–919. Takahashi, K., Honda, H., Akiba, Y., Horiguchi, M., 1992. Effects of dietary protein and corticosterone on mixed function oxidase in liver and immunity in male broilers. Jpn. Poult. Sci. 29, 350–360. Totzke, U., Fenske, M., Hüppop, O., Raabe, H., Schach, N., 1999. The influence of fasting on blood and plasma composition of herring gulls (Larus argentatus). Physiol. Biochem. Zool. 72, 426–437. Van Noordwijk, A.J., de Jong, G., 1986. Acquisition and allocation of rescources: their influence on variation in life history tactics. Am. Nat. 128, 137–141. Zhou, H., Buitenhuis, A., Weigend, S., Lamont, S., 2001. Candidate gene promoter polymorphisms and antibody response kinetics in chickens: interferon-gamma, interleukin-2, and immunoglobulin light chain. Poult. Sci. 80, 1679–1689.