Growing altricial birds may experience nutritional stress in the nest due to sibling competition, food restriction, or parasites. Nutritional stress may be detected through its effect on nestling growth, although genetic and maternal effects may interfere with its expression. A more direct way of estimating nutritional stress may be through measurements of organismic stress in growing birds. Heterophil/lymphocyte ratios (H/L) have been proposed as reliable physiological indicators of the stress response. Heat-shock protein (HSP) synthesis is induced by animals in response to various stressors, making the concentration of HSPs a suitable measure of organismic stress. In a study of growing pied flycatchers (Ficedula hypoleuca), we assessed H/L from cell counts in blood smears and levels of two HSPs, HSP60 and HSP70, from the cell fraction of peripheral blood. H/L and the level of HSP60 were significantly positively associated in nestlings of 13 days of age, while the level of HSP70 was not related to the other two measures. Small nestlings with respect to tarsus length on day 7 had higher levels of HSP60 and higher H/L values. Brood means of nestling mass, tarsus length, and wing length shortly before fledging were negatively related to both mean HSP60 level and mean H/L ratio. Brood variances in body mass and wing length on day 13 were positively associated with mean HSP60 levels but not with mean H/L values. Up to 80% of variation in some traits was explained by stress indicators.

Summary: This study presents an easy protocol to measure the amount of immunoglobulins from the blood serum of different bird species in the wild (Ficedula hypoleuca Pallas, Parus caeruleus L., Lanius meridionalis Temminkck, Lanius collurio L., Athene noctua Scopoli and Falco tinnunculus L.) by direct enzyme‐linked immunosorbent assay, ELISA using commercial antichicken antibodies. Additionally, the ELISA technique is validated for detecting serum immunoglobulins by means of other electrophoretic (sodium dodecyl sulphate polyacrylamide gel electrophoresis, SDS‐PAGE, and native electrophoresis) and immunological (Western blot) methods. The results by Western blot show that the commercial antibody recognized proteins with apparent molecular weight according to heavy and light chains of immunoglobulins. Both ELISA and Western blot data were correlated, implying that the commercial antibody bound to immunoglobulins and not to other proteins or ELISA plates. Densitometric data achieved by SDS‐PAGE and native electrophoresis were only correlated in some species indicating a problem in detecting clearly the heavy and light chains, and γ‐globulin fraction, respectively. It is concluded that the proposed protocol is easy to carry out and may be used to detect total serum immunoglobulins from most bird species.

SUMMARY: AvianPlasmodiumandHaemoproteusparasites are easily detected by DNA analyses of infected samples but only correctly assigned to each genus by sequencing and use of a phylogenetic approach. Here, we present a restriction site to differentiate between both parasite genera avoiding the use of those analyses. Alignments of 820 sequences currently listed in GenBank encoding a particular cytochrome B region of avianPlasmodiumandHaemoproteusshow a shared restriction site for both genera using the endonucleaseHpyCH4III. An additional restriction site is present inPlasmodiumsequences that would initially allow differentiation of both genera by differential migration of digested products on gels. Overall 9 out of 326 sequences containing both potential restriction sites do not fit to the general rule. We used this differentiation of parasite genera based onHpyCH4III restriction sites to evaluate the efficacy of 2 sets of general primers in detecting mixed infections. To do so, we used samples from hosts infected by parasites of both genera. The use of general primers was only able to detect 25% or less of the mixed infections. Therefore, parasite DNA amplification using general primers to determine the species composition of haemosporidian infections in individual hosts is not recommended. Specific primers for each species and study area should be designed until a new method can efficiently discriminate both parasites.