Sex-specific effects of handling time on an index of immune activity in zebra finches

Recently, there has been considerable interest in the role of the immune system in shaping life-history evolution, sexual selection strategies, and indexes of individual quality. The most frequently used assay of immune function, particularly in avian field studies, is the phytohemagglunitin (PHA) skin test. PHA is injected subcutaneously into the wing web, and the magnitude of the resultant swelling has traditionally been interpreted as an index of an individual's cell-mediated immunocompetence. The test follows one of two protocols: the traditional two-wing injection protocol, with one wing web injected with PHA and the other with phosphate-buffered saline (PBS), or the simplified one-wing protocol that omits the PBS injection. In this technical comment, we alert researchers to the importance of considering handling time when performing the PHA test. We show that zebra finches (Taeniopygia guttata) subjected to the two-wing protocol had a lower wing-web swelling than individuals injected in one wing. In males, handling time explained over 50% of the variation in an individual's skin swelling response; females were relatively unaffected by handling time. We suggest that caution should be exercised when comparing the magnitude of wing-web swelling across studies in which the alternate protocol was followed. In addition, the recording of handling time, and its inclusion in subsequent statistical analyses, may aid in the detection of subtle differences across treatments.     

Reproductive state, but not testosterone, reduces immune function in male house sparrows (Passer domesticus)

The immune system requires energetic and nutritional resources to optimally defend organisms against pathogens and parasites. Because resources are typically limited, immune function may require a trade-off with other physiologically demanding activities. Here, we examined whether photoperiodically induced seasonal states (breeding, molting, or nonbreeding) affected the cutaneous immune response of captive male house sparrows (Passer domesticus). To assess immune function in these birds, we injected the mitogen phytohemagglutinin (PHA) into the patagium and measured the resulting wing web swelling. Molting and nonbreeding birds had similar immune responses to PHA injection. However, males in a breeding state showed lower immune responses than both molting and nonbreeding birds even though they did not actually breed. We tested whether this decrease in the PHA swelling response in birds in a breeding state was due to elevated plasma concentrations of testosterone (T) by administering T to birds in a nonbreeding state. Contrary to some evidence in the literature, T did not suppress the response to PHA in house sparrows. Our data show that passerine birds show seasonal modulation in immune function, even in benign environmental conditions. However, even though T is often cited as a strong immunosuppressant, it is not fully responsible for this seasonal modulation.     

No effect of blood sampling or phytohaemagglutinin injection on postfledging survival in a wild songbird

The injection of phytohaemagglutinin (PHA) and sampling of blood are widely used in studies of wild vertebrates to assess components of immune and endocrine function and health state and to obtain genetic material. Despite the pervasive use of these techniques in the life sciences, their potential effects on survival are rarely considered. For example, whether injection of the immunogen PHA into body parts critical for locomotion (e.g., the prepatagium, or wing web, in birds) affects survival has not been tested. Here, we test whether injection of PHA into the wing web and blood sampling from nestling house wrens affects their subsequent recruitment and survival as breeding adults. Capture‐mark‐recapture analysis on a large sample of young (N = 20,152 fledglings from 3959 broods) treated over 10 years revealed that neither PHA injection nor blood sampling affected individual survival and detection probability. Recruitment as a breeder varied among years, but this variation was not attributable to sampling effort, or the percent of all adults identified at the nest during a given year. Variation in the percent of adults identified was primarily attributable to the effect of nest depredation on our ability to capture nesting pairs. Our results indicating lack of an effect of blood sampling and immune stimulation on survival are encouraging, but we recommend further work to assess the potential negative effects of all commonly used techniques on the survival of study subjects in the wild, including the potential costs associated with mounting various immunological responses.     

Immune activity elevates energy expenditure of house sparrows: a link between direct and indirect costs?

The activation of an immune response is beneficial for organisms but may also have costs that affect fitness. Documented immune costs include those associated with acquisition of special nutrients, as well as immunopathology or autoimmunity. Here, we test whether an experimental induction of the immune system with a non-pathological stimulant can elevate energy turnover in passerine birds. We injected phytohaemagglutinin (PHA), a commonly used mitogen that activates the cell-mediated immune response, into the wing web of house sparrows, Passer domesticus. We then examined energetic costs resulting from this immune activity and related those costs to other physiological activities. We found that PHA injection significantly elevated resting metabolic rate (RMR) of challenged sparrows relative to saline controls. We calculated the total cost of this immune activity to be ca. 4.20 kJ per day (29% RMR), which is equivalent to the cost of production of half of an egg (8.23 kJ egg(-1)) in this species. We suggest that immune activity in wild passerines increases energy expenditure, which in turn may influence important life-history characteristics such as clutch size, timing of breeding or the scheduling of moult.     

Secondary phytohaemagglutinin (PHA) swelling response is a good indicator of T‐cell‐mediated immunity in free‐living birds

The validity of using the phytohaemagglutinin (PHA) test to measure acquired immunity, one of the most widely used methods, is currently being debated due to new knowledge on the complex physiology of the process. As a greater secondary response to repeated challenges linked to increases of circulating lymphocyte levels would be indicative of a T‐cell‐mediated immune response, we performed for the first time an experiment under natural conditions with repeated PHA challenges in free‐living adult birds and chicks to shed light on this topic. We found significantly stronger secondary response to PHA injection independent of sex or age, while controlling for body condition, the second response being on average 90% larger than the first. Likewise, lymphocyte counts were significantly higher in the second PHA challenge, whereas no significant differences were found among untreated birds. Significant positive correlations between the PHA response and both lymphocyte counts and plasma protein levels (mainly albumin, globulin precursor) were recovered, whereas no significant differences were recovered in plasma protein levels between challenges. Our results are consistent with those from captive birds, supporting the validity of the PHA skin‐swelling test as an accurate gauge of acquired T‐cell‐mediated immunity in birds.     

Social environment and immunity in male red jungle fowl

We examined the relationship between social dominance, immuneresponse, and ornamentation in captive red jungle fowl by comparingthese variables in males housed individually with a singlefemale to those in the same males after they were placed inflocks with an unfamiliar male and three unfamiliar females. Males with larger combs before being placed in the flocks weremore likely to become dominant, and dominant males' combs grewafter flock formation, whereas subordinate males' combs shrank.Immune response as reflected in hematocrit, immunoglobulinlevels, and wing web swelling (a measure of cell-mediated immunity)was stronger in males that later became dominant, both beforeand after flock formation, although the difference betweendominant and subordinate birds was more pronounced after maleswere housed in the multi-male groups. Dominant and subordinatemales also differed in the relationship between comb lengthand wing web swelling. Among dominant males, individuals withlarger combs had significantly larger swellings after flock formation, whereas within the subordinate males, those withrelatively larger combs had worse cell-mediated immunity thanthose with smaller combs. These results suggest that malesof different quality pay different costs to maintain both ornamentationand immune defense.     

Cytokine expression in phytohaemagglutinin‐induced skin inflammation in a galliform bird

Researchers interested in ecological immunology face substantial methodological problems: 1) most immunological approaches are difficult to perform in free‐living animals, 2) in some of the applicable methods the immunological background of the test remains unclear. The latter is also true for the phytohaemagglutinin (PHA) skin‐swelling test, a trait of cell‐mediated immunity commonly measured in ecology. A lack of direct evidence documenting the immunological processes in the tissue limits our understanding of the mechanism triggering the response to PHA. Understanding of this mechanism is, nonetheless, crucial for us to uncover the nature of ecological costs and benefits of investments into the response. As knowledge of cytokine signalling in the tissue may clarify the response mechanism, in our study we investigated the association between the PHA‐induced skin‐swelling and tissue cytokine expression in males of grey partridge Perdix perdix. In PHA‐challenged birds we assessed expression of nine cytokines (IL‐1β, IL‐2, IL‐4, IL‐6, IL‐10, IL‐12, IL‐17, TGF‐β, IFN‐γ) in wing‐web skin during an early stage of the immune response. We examined the relationship between the magnitude of tissue swelling and cytokine expression. Contrary to some earlier expectations we did not find any differential expression of T‐cell growth factor, IL‐2, in the tissue. Hence, T‐cell proliferation at the time of the swelling measurement is unlikely. We detected differential expression in Th17 pro‐inflammatory (IL‐1β, IL‐6) and anti‐inflammatory (TGF‐β) cytokines. The PHA‐induced swelling response was only weakly linked to the expression of TGF‐β. We also found relationships between the PHA‐induced swelling response and phenotypic traits of the birds; the PHA swelling was positively associated with the extent of melanin‐based breast ornamentation and negatively related to body size. Our results might suggest that variation in swelling is influenced by total numbers of responding cells rather than by differences in signalling. Moreover, we revealed significant correlations in expression of IL‐1β, IL‐6 and TGF‐β. These findings are the first to show on the molecular level that the PHA skin‐swelling test actually measures inflammation process which is part of innate immune defence and not the adaptive immune response (as assumed if the test was the reflection of T‐cell proliferation).     

不同繁殖状态雌性黑线仓鼠对植物血凝素的反应模式

由植物血凝素（Phytohemagglutinin,ＰＨＡ） 诱导产生的肿胀反应是生态免疫学中被广泛使用的免疫学参数,通过注射部位的肿胀程度大体上可反映细胞介导的免疫和天然免疫的综合变化情况。为探讨小型哺乳动物不同繁殖状态对ＰＨＡ 的反应模式,以非繁殖期、妊娠期、哺乳期和断乳期的雌性黑线仓鼠为研究对象,测定了注射ＰＨＡ 和生理盐水前（０ ｈ）及注射后６ ｈ、１２ ｈ 和２４ ｈ 注射部位皮肤组织的增厚程度。结果显示：（１）黑线仓鼠对ＰＨＡ 的反应有两种模式：非繁殖期、妊娠期和断乳期对ＰＨＡ 的反应模式相似,都在注射后６ ｈ 最高,１２ ｈ 和２４ ｈ 后逐渐下降;哺乳期对ＰＨＡ 的反应在注射后６ ｈ、１２ ｈ 和２４ ｈ 接近,彼此间的差异均不显著。（２）哺乳期黑线仓鼠的ＰＨＡ 反应高峰值有下降趋势,但与非繁殖期、妊娠期和断乳期高峰值之间的差异不显著。结果表明,黑线仓鼠在哺乳期对ＰＨＡ 的反应模式不同于其他繁殖阶段,且反应的高峰值被延迟,这对其存活可能有害,但有助于繁殖进程的延续和后代的生长发育。     

Sex differences in circulating antibodies in nestling Pied Flycatchers Ficedula hypoleuca

Sex differences in immune function are relatively well studied in vertebrate animals, although the patterns are not always clear in birds. The study of immune responses in nestlings of wild bird populations may constitute an appropriate way to investigate inherent intersexual differences while controlling for environmental conditions such as parasitism that affect male and female individuals growing in the same nest. We studied whether the cell‐mediated immune response, as measured by phytohaemagglutinin (PHA) injection, and the levels of circulating antibodies differ between sexes of Pied Flycatcher nestlings Ficedula hypoleuca. No sex differences in nestling cell‐mediated immune response were found, but females showed significantly higher levels of plasma immunoglobulins than males did. Although nestling birds may not have a fully functional humoral immune defence, our study indicates that sex differences in the humoral component exist at this early stage of life. Given the importance of antibodies in the fight against parasite, bacterial and viral infections, the intrinsic sex disparity in circulating antibodies may have important implications for the life history of each sex.     

Immunity to transplantable carcinogen induced fibrosarcomas in B2/B2 chickens. II. Macrophage dependency of adoptive T cell mediated tumor immunity.

The nature of immunity to transplantable chemically induced fibrosarcomas in SC (B2/B2) chicken was examined using Winn tests performed in the wing web. Immunity in spleen cell donors was induced by pretreatment with C. parvum or BCG followed by tumor cells + bacterial adjuvant in one and tumor cells alone in the other wing web. The T cells mediating the adoptive immunity were sensitive to anti-T + C, nylon wool nonadherent, mitomycin resistant and radiation (1000 R) sensitive. The adoptive immunity could not be expressed in heavily irradiated recipients or in hosts pretreated with trypan blue or silica. The host contribution could be reconstituted by iv injection of spleen or bone marrow cells from agamma-globulinemic (Aγ) unimmunized donors 2 days prior to Winn tests or by the local injection into wing webs of spleen cells or purified peripheral blood monocytes from Aγ donors. It was concluded that cooperation between immune donor T cells and normal monocytes of host origin mediated the inhibition of SCFS growth in Winn tests.