The diversity of the cloacal microbial community in migratory shorebirds, caught at the Tagus estuary, Portugal, was assessed by cultivation (R2A and Nutrient Agar media) and denaturing gradient gel electrophoresis profiling (DGGE) to provide a better understanding of the birds' potential to harbor and disperse pathogens. Three different bird species belonging to four different populations were studied: common redshank (Tringa totanus), black-winged stilt (Himantopus himantopus) and nominate and Icelandic populations of black-tailed godwit (Limosa limosa). DGGE profiling and partial 16S RNA gene sequences of 240 isolates, and 26 DGGE bands resulting in 58 clones, were analyzed. Most isolates were members of the phylum Firmicutes and Actinobacteria and only a small portion belonged to the Proteobacteria and DeinococcusThermus phyla. Potentially pathogenic strains carried by the birds were found such as Helicobacter and Staphylococcus in all bird species, and Clostridium, Mycobacterium, Rhodococcus, Legionella and Corynebacterium in black-winged stilts. Unexpectedly, bacteria from the phylum Deinococcus-Thermus were isolated in shorebirds and were present in all the bird species studied.

SummaryMigratory wader populations face global threats, mainly related to increasing rates of habitat loss and disturbance driven by human activities. To a large extent, the long-term survival of these populations requires the conservation of networks of sites along their migratory flyways. The Tagus estuary, Portugal, is among the most important wetlands for waders in the East Atlantic Flyway. Annual winter wader counts have been carried in this wetland since 1975 and a monthly roost-monitoring programme was implemented in 2007. Wintering populations of three out of the five most abundant species, Dunlin Calidris alpina, Grey Plover Pluvialis squatarola and Redshank Tringa totanus, showed significant population declines over the past three decades, which are most likely due to the loss and degradation of roost sites as a result of increasing human activity. The situation is unlikely to improve, as a high proportion of the wintering waders use roost sites that are situated in highly urbanised areas with no legal protection. The use of different roost sites by waders is highly variable both temporally and spatially, thus emphasizing the need for a network of good quality roost sites. Additionally, during migration, 60-80% of all the waders of the Tagus estuary concentrate at a single refuge, thus increasing the risk for wader populations during these periods. SumárioAs populações de aves limícolas migradoras enfrentam ameaças globais, maioritariamente relacionadas com a crescente perda de habitat e perturbação humana. Para garantir a sobrevivência a longo-termo destas populações é imprescindível a conservação de uma rede de áreas húmidas ao longo das suas rotas migratórias. O estuário do Tejo, em Portugal, é uma das mais importantes áreas húmidas para aves limícolas na rota Migratória do Atlântico Este. Desde 1975, foram efectuadas contagens anuais de aves limícolas no inverno nesta zona húmida e, em 2007, foi implementado um programa de monitorização mensal dos refúgios de preia-mar. As populações invernantes de três das cinco espécies de limícolas mais abundantes no estuário, o Pilrito-de-peito-preto Calidris alpina, a Tarambola-cinzenta Pluvialis squatarola e o Perna-vermelha Tringa totanus, mostraram tendências significativas de decréscimo nas últimas três décadas, potencialmente devido à degradação e perda de refúgios como resultado da crescente actividade antropogénica. Esta situação poderá deteriorar-se, uma vez que uma grande proporção das aves limícolas invernantes utiliza refúgios em áreas densamente urbanizadas sem qualquer estatuto legal de protecção. A utilização dos diferentes refúgios do estuário do Tejo por aves limícolas tem uma grande variação espacial e temporal, o que reforça a importância da existência de uma rede de refúgios de elevada qualidade nesta área húmida. Durante a migração, 60 a 80% das aves limícolas concentram-se num único refúgio, ficando assim particularmente vulneráveis neste período.

BackgroundOver the past couple of decades, measuring immunological parameters has become widespread in studies of ecology and evolution. A combination of different immunological indices is useful for quantifying different parts of the immune system and comprehensively assessing immune function. Running multiple immune assays usually requires samples to be repeatedly thawed and re-frozen. There is some evidence that repeated freezing and thawing can affect assay results, but this has never been comprehensively studied in some common ecological immunology assays. We tested the effect of multiple (1, 2, 3, 4, 5, 10) freeze-thaw cycles on the results of four commonly used immunological assays: haemolysis-haemagglutination titres, haptoglobin concentration, bacterial killing capacity and total immunoglobulins (IgY). We tested five different bird species from four different bird orders (Passeriformes, Columbiformes, Charadriiformes and Galliformes), and we included both captive and free-living individuals. In addition, we tested for haptoglobin concentrations and the haemolysis-haemagglutination assay if re-analysing samples 1 year apart led to different results. For the haemolysis-haemagglutination assay we also tested two different sources of rabbit blood, and we compared untreated microtitre plates with plates that were “blocked” to prevent nonspecific interactions between the plate and assay reagents.ResultsRepeated freezing and thawing of plasma had no effect on lysis titres, haptoglobin concentrations, bacterial killing capacity, or total immunoglobulin levels. Agglutination titres were unaffected by up to five cycles but were lower after ten freeze-thaw cycles. For the haemolysis-haemagglutination assay and haptoglobin concentrations, re-analysing samples 1 year apart yielded highly correlated data. For the haemolysis-haemagglutination assay, the source of rabbit blood did not influence the results, and the untreated vs. blocked plates differed slightly overall, but at the individual level assay results were highly correlated. Using different rabbit blood sources or different types of microtitre plates yielded highly correlated data.ConclusionsOur data suggest that repeated freeze-thaw cycles do not impair assay results to the point of influencing ecological or evolutionary conclusions. Plasma samples can be safely stored in one tube and thawed repeatedly for different assays. Nevertheless, we recommend consistent treatment of samples in terms of freeze-thaw cycles or other laboratory treatments to minimize the potential for introducing a systematic bias.

In their African freshwater wintering habitats, shorebirds show a high prevalence of blood parasites, whereas no parasites are detected elsewhere along the migration route. We looked at two genera of haemosporidian parasites, Haemoproteus and Plasmodium, in the long-distance migrating Ruff (Philomachus pugnax) along a geographical/seasonal gradient to verify the infection pattern and examine possible hidden organ infections at European staging areas. We amplified parasite DNA from blood of 53 healthy birds wintering in Mali, 53 samples of seven organ tissues (spleen, liver, kidneys, heart, lungs, brain, and pectoral muscle) from healthy individuals caught during spring migration, and 18 weak birds found sick in summer in The Netherlands. We confirm that Ruffs wintering in Africa carried blood infections and that some infections developed into hidden organ infections during spring migration. Moreover, sick birds either had new infections (in one juvenile) or relapses (in an adult harboring an African lineage). Our results suggest that some parasites develop latency. This strategy may be beneficial for the parasite as it may take control over reappearance in the blood to help further transmission.

The host specificity and host sharing of avian haemoparasites (genera Haemoproteus and Plasmodium) is still poorly known, although they infect a large proportion of several studied bird populations. This study used molecular techniques to detect haemoparasites in marsh warblers and in other passerines that feed in reed beds, at 4 sites in Portugal. The host-specificity of the parasite lineages was analysed and compared with other cases described in the literature to assess whether apparent host specificity changes according to the studied system. Nine lineages of Haemoproteus and 15 of Plasmodium were found, of which only 10 Plasmodium were proven to have local transmission. Each lineage was confined to a distinct set of host species. The distribution of parasites in the host species was non-nested, meaning that specialist lineages did not always share hosts with generalists. The most prevalent lineages were those with a wider host range, indicating that the ability to infect more hosts will enhance a parasite's prevalence in its entire host range. We also found that in our areas, a specialist parasite (H. MW1) appears to have a more generalist character than described in the literature, suggesting that a parasite's apparent specialization can depend on the type of host species sampled.

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The major histocompatibility complex (MHC) encodes proteins that are central for antigen presentation and pathogen elimination. MHC class I (MHC-I) genes have attracted a great deal of interest among researchers in ecology and evolution and have been partly characterized in a wide range of bird species. So far, the main focus has been on species within the bird orders Galliformes and Passeriformes, while Charadriiformes remain vastly underrepresented with only two species studied to date. These two Charadriiformes species exhibit striking differences in MHC-I characteristics and MHC-I diversity. We therefore set out to study a third species within Charadriiformes, the Icelandic subspecies of black-tailed godwits (Limosa limosa islandica). This subspecies is normally confined to parasite-poor environments, and we hence expected low MHC diversity. MHC-I was partially characterized first using Sanger sequencing and then using high-throughput sequencing (MiSeq) in 84 individuals. We verified 47 nucleotide alleles in open reading frame with classical MHC-I characteristics, and each individual godwit had two to seven putatively classical MHC alleles. However, in contrast to previous MHC-I data within Charadriiformes, we did not find any evidence of alleles with low sequence diversity, believed to represent non-classical MHC genes. The diversity and divergence of the godwits MHC-I genes to a large extent fell between the previous estimates within Charadriiformes. However, the MHC genes of the migratory godwits had few sites subject to positive selection, and one possible explanation could be a low exposure to pathogens.Electronic supplementary materialThe online version of this article (doi:10.1007/s00251-017-0993-7) contains supplementary material, which is available to authorized users.