Backgroundcological immunology requires techniques to reliably measure immunocompetence in wild vertebrates. The PHA-skin test, involving subcutaneous injection of a mitogen (phytohemagglutinin, PHA) and measurement of subsequent swelling as a surrogate of T-cell mediated immunocompetence, has been the test of choice due to its practicality and ease of use in the field. However, mechanisms involved in local immunological and inflammatory processes provoked by PHA are poorly known, and its use and interpretation as an acquired immune response is currently debated.MethodologyHere, we present experimental work using a variety of parrot species, to ascertain whether PHA exposure produces larger secondary than primary responses as expected if the test reflects acquired immunocompetence. Moreover, we simultaneously quantified T-lymphocyte subsets (CD4+, CD5+ and CD8+) and plasma proteins circulating in the bloodstream, potentially involved in the immunological and inflammatory processes, through flow cytometry and electrophoresis.Principal FindingsOur results showed stronger responses after a second PHA injection, independent of species, time elapsed and changes in body mass of birds between first and second injections, thus supporting the adaptive nature of this immune response. Furthermore, the concomitant changes in the plasma concentrations of T-lymphocyte subsets and globulins indicate a causal link between the activation of the T-cell mediated immune system and local tissue swelling.Conclusions/SignificanceThese findings justify the widespread use of the PHA-skin test as a reliable evaluator of acquired T-cell mediated immunocompetence in diverse biological disciplines. Further experimental research should be aimed at evaluating the relative role of innate immunocompetence in wild conditions, where the access to dietary proteins varies more than in captivity, and to ascertain how PHA responses relate to particular host-parasite interactions.

Individual quality is often signaled by phenotypic flags, such as bright plumage patches in birds. Extended phenotype signals can similarly show quality, but in these cases the signals are external to the individual, often taking the form of objects scavenged from the environment. Through multiple manipulative experiments, we showed that objects used for nest decoration by a territorial raptor, the black kite (Milvus migrans), act as reliable threats to conspecifics, revealing the viability, territory quality, and conflict dominance of the signaler. Our results suggest that animal-built structures may serve as signaling devices much more frequently than currently recognized.

Antibiotic residues that may be present in carcasses of medicated livestock could pass to and greatly reduce scavenger wildlife populations. We surveyed residues of the quinolones enrofloxacin and its metabolite ciprofloxacin and other antibiotics (amoxicillin and oxytetracycline) in nestling griffon Gyps fulvus, cinereous Aegypius monachus and Egyptian Neophron percnopterus vultures in central Spain. We found high concentrations of antibiotics in the plasma of many nestling cinereous (57%) and Egyptian (40%) vultures. Enrofloxacin and ciprofloxacin were also found in liver samples of all dead cinereous vultures. This is the first report of antibiotic residues in wildlife. We also provide evidence of a direct association between antibiotic residues, primarily quinolones, and severe disease due to bacterial and fungal pathogens. Our results indicate that, by damaging the liver and kidney and through the acquisition and proliferation of pathogens associated with the depletion of lymphoid organs, continuous exposure to antibiotics could increase mortality rates, at least in cinereous vultures. If antibiotics ingested with livestock carrion are clearly implicated in the decline of the vultures in central Spain then it should be considered a primary concern for conservation of their populations.

The impact on wildlife health of the increase in the use of antimicrobial agents with the intensification of livestock production remains unknown. The composition, richness and prevalence of cloacal microflora as well as bacterial resistance to antibiotics in nestlings and full-grown Egyptian vultures Neophron percnopterus were assessed in four areas of Spain in which the degree of farming intensification differs. Differences in diet composition, especially the role of stabled livestock carrion, appear to govern the similarities of bacterial flora composition among continental populations, while the insular vulture population (Fuerteventura, Canary Islands) showed differences attributed to isolation. Evidence of a positive relationship between the consumption of stabled livestock carrion and bacterial resistance to multiple antibiotics was found. Bacterial resistance was high for semisynthetic penicillins and enrofloxacin, especially in the area with the most intensive stabled livestock production. The pattern of antibiotic resistance was similar for the different bacterial species within each area. Bacterial resistance to antibiotics may be determined by resistance of bacteria present in the livestock meat remains that constituted the food of this species, as indicated by the fact that resistance to each antibiotic was correlated in Escherichia coli isolated from swine carrion and Egyptian vulture nestlings. In addition, resistance in normal faecal bacteria (present in the microflora of both livestock and vultures) was higher than in Staphylococcus epidermidis, a species indicator of the transient flora acquired presumably through the consumption of wild rabbits. Potential negative effects of the use of antimicrobials in livestock farming included the direct ingestion of these drug residues and the effects of bacterial antibiotic resistance on the health of scavengers.

Pathogen diversity is thought to drive major histocompatibility complex (MHC) polymorphism given that host's immune repertories are dependent on antigen recognition capabilities. Here, we surveyed an extensive community of pathogens (n = 35 taxa) and MHC diversity in mainland versus island subspecies of the Eurasian kestrel Falco tinnunculus and in a sympatric mainland population of the phylogenetically related lesser kestrel Falco naumanni. Insular subspecies are commonly exposed to impoverished pathogen communities whilst different species' ecologies and contrasting life-history traits may lead to different levels of pathogen exposure. Although specific host traits may explain differential particular infections, overall pathogen diversity, richness and prevalence were higher in the truly cosmopolitan, euriphagous and long-distance disperser Eurasian kestrel than in the estenophagous, steppe-specialist, philopatric but long-distance migratory lesser kestrel. Accordingly, the continental population of Eurasian kestrels displayed a higher number (64 vs. 49) as well as more divergent alleles at both MHC class I and class II loci. Detailed analyses of amino acid diversity revealed that significant differences between both species were exclusive to those functionally important codons comprising the antigen binding sites. The lowest pathogen burdens and the smallest but still quite divergent set of MHC alleles (n = 16) were found in island Eurasian kestrels, where the rates of allele fixation at MHC loci seem to have occurred faster than at neutral markers. The results presented in this study would therefore support the role of pathogen diversity and abundance in shaping patterns of genetic variation at evolutionary relevant MHC genes.

Insular populations have attracted the attention of evolutionary biologists because of their morphological and ecological peculiarities with respect to their mainland counterparts. Founder effects and genetic drift are known to distribute neutral genetic variability in these demes. However, elucidating whether these evolutionary forces have also shaped adaptive variation is crucial to evaluate the real impact of reduced genetic variation in small populations. Genes of the major histocompatibility complex (MHC) are classical examples of evolutionarily relevant loci because of their well-known role in pathogen confrontation and clearance. In this study, we aim to disentangle the partial roles of genetic drift and natural selection in the spatial distribution of MHC variation in insular populations. To this end, we integrate the study of neutral (22 microsatellites and one mtDNA locus) and MHC class II variation in one mainland (Iberia) and two insular populations (Fuerteventura and Menorca) of the endangered Egyptian vulture (Neophron percnopterus). Overall, the distribution of the frequencies of individual MHC alleles (n=17 alleles from two class II B loci) does not significantly depart from neutral expectations, which indicates a prominent role for genetic drift over selection. However, our results point towards an interesting co-evolution of gene duplicates that maintains different pairs of divergent alleles in strong linkage disequilibrium on islands. We hypothesize that the co-evolution of genes may counteract the loss of genetic diversity in insular demes, maximize antigen recognition capabilities when gene diversity is reduced, and promote the co-segregation of the most efficient allele combinations to cope with local pathogen communities.

Summary 1.Despite the important role of refuse dumps for the conservation of threatened avian scavengers, there is no information about the direct effects on scavenger health from feeding on meat remains found at these places, including the potential for exposure to infections agents and harmful chemical residues. 2.We assessed the composition, species richness and prevalence of faecal microflora of wintering red kites Milvus milvus in two areas of central Spain where kites fed primarily on carrion from stabled livestock reared in intensive conditions (Segovia) versus those that fed on wild rabbits Oryctolagus cuniculus (Madrid). 3. Faecal bacterial flora composition, species richness and prevalence differed between areas. These differences were attributed to diet and the environmental conditions found at the sites where the kites obtained most of their food. The intestinal microflora of kites feeding on carrion from stabled livestock (mostly fattening pigs) differed from the flora considered to be normal for a healthy raptor. 4. Salmonella sp. was found in a relatively high proportion of samples from Segovia (25%), 6% of those samples yielded in pure culture (serotypes enteritidis and gallinarum ). If these serotypes cause disease in kites and other raptors, as they do for poultry and some other birds, there may be cause for concern regarding the health of an important sector of the red kite wintering population. 5. Synthesis and applications . Conservation of wintering red kites in Spain should involve management to promote the recovery of populations of wild prey, especially wild rabbits, and measures to allow free-range livestock carcasses to be placed in the countryside for scavenger elimination without posing a risk from the spread and transfer of disease, especially bovine spongiform encephalopathy (BSE). Periodic inspections by management agencies should ensure that meat originating from stabled livestock and disposed of in refuse dumps does not pose major hazards for avian scavengers. The risk to scavengers from ingestion of residues of veterinary drugs, pathogen acquisition and gut flora alteration, arising from disposal and accumulation of infected carcasses and rotten meat in refuse dumps, must be carefully monitored and managed.

Competition for resources (e.g. mates or food) is the main evolutionary explanation for conspicuous ornaments in males, although this idea is not generalized in females. Whether or not the expression of melanic coloration is dependent on environmental conditions remains controversial. We studied three different melanin-based female traits in the Eurasian kestrel Falco tinnunculus, a sexually dichromatic species, for a period of 10 years: grey coloration in rump and tail and the width of the black subterminal tail band. We analysed these traits for within-individual variation among years, as well as their possible link with indices of quality, such as age, body size, and breeding performance. The results obtained demonstrate that female melanin-based coloration increased from yearlings to adults. In addition, the expression of female rump coloration covaried positively with the environmental conditions in the previous year (i.e. measured as clutch size at population level). Finally, we found a positive correlation between grey rump coloration and clutch size. These results suggest that the expression of rump coloration, a melanin-based trait, is environmentally constrained, and we propose that this character could function as an indicator of individual quality in female Eurasian kestrels.