Search

The major histocompatibility complex (MHC) hosts the most polymorphic genes ever described in vertebrates. The MHC triggers the adaptive branch of the immune response, and its extraordinary variability is considered an evolutionary consequence of pathogen pressure. The last few years have witnessed the characterization of the MHC multigene family in a large diversity of bird species, unraveling important differences in its polymorphism, complexity, and evolution. Here, we characterize the first MHC class II B sequences isolated from a Rallidae species, the Eurasian Coot Fulica atra. A next-generation sequencing approach revealed up to 265 alleles that translated into 251 different amino acid sequences (β chain, exon 2) in 902 individuals. Bayesian inference identified up to 19 codons within the presumptive peptide-binding region showing pervasive evidence of positive, diversifying selection. Our analyses also detected a significant excess of high-frequency segregating sites (average Tajima's D = 2.36, P < 0.05), indicative of balancing selection. We found one to six different alleles per individual, consistent with the occurrence of at least three MHC class II B gene duplicates. However, the genotypes comprised of three alleles were by far the most abundant in the population investigated (49.4%), followed by those with two (29.6%) and four (17.5%) alleles. We suggest that these proportions are in agreement with the segregation of MHC haplotypes differing in gene copy number. The most widespread segregating haplotypes, according to our findings, would contain one single gene or two genes. The MHC class II of the Eurasian Coot is a valuable system to investigate the evolutionary implications of gene copy variation and extensive variability, the greatest ever found, to the best of our knowledge, in a wild population of a non-passerine bird.

Teschovirus and Sapelovirus are two genera of the Picornaviridae family, comprising highly variable and heterogeneous enteric viruses, commonly found in faecal samples from domestic pigs. Although both of them are also known to infect wild boar, studies on their presence in these wild suids are scarce. The present study aimed at determining the presence of porcine teschovirus (PTV) and sapelovirus (PSV) in free-living wild boar populations, as well as to study their relationships with similar viruses present in pigs. Fresh faecal samples (n=63) from wild boar were collected in Doñana Biological Reserve (SW Spain) during 2007 and 2011, and analysed using multiplex RT-PCR for the simultaneous detection and differentiation of PTV and PSV. A total of 32 samples (50.8%) presented positive PTV bands, while PSV amplicons were detected in 4 samples (6.4%). All PSV-positive samples were also positive for PTV, which indicated co-infection with both viruses. Virus isolation was successful from 6 samples, 4 of which were identified as PTV by RT-PCR, and three of these were further characterized by sequencing of the VP1 capsid protein. The remaining two isolates were negative for PTV or PSV. Genetic characterization of PSV-positive faecal samples, using the VP4 protein coding gene, was successful in 4 stool samples. Close phylogenetic relationship was found among wild boar and domestic pig strains in both PTV and PSV. More studies are needed to ascertain the epizootiological significance of these findings., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Background: Blood-sucking flying insects play a key role in the transmission of pathogens of vector-borne diseases. However, at least for the case of avian malaria parasites, the vast majority of studies focus on the interaction between parasites and vertebrate hosts, but there is a lack of information regarding the interaction between the parasites and the insect vectors. Here, we identified the presence of malaria and malaria-like parasite lineages harbored by the potential vector Culicoides circumscriptus (Kieffer). Also, we identified some nodes of the transmission network connecting parasite lineages, potential insect vectors and avian hosts by comparing Haemoproteus and Plasmodium lineages isolated from insects with those infecting wild birds in this and previous studies., Methods: Using a molecular approach, we analysed the presence of blood parasites in a total of 97 biting midges trapped in the Doñana National Park (SW Spain) and surrounding areas. Also, 123 blood samples from 11 bird species were analyzed for the presence of blood parasite infections. Blood parasites Haemoproteus and Plasmodium were identified by amplification of a 478 bp fragment of the mitochondrial cytochrome b gen., Results: Thirteen biting midges harboured blood parasites including six Haemoproteus and two Plasmodium lineages, supporting the potential role of these insects on parasite transmission. Moreover, ten (8.1%) birds carried blood parasites. Seven Plasmodium and one Haemoproteus lineages were isolated from birds. Overall, six new Haemoproteus lineages were described in this study. Also, we identified the transmission networks of some blood parasites. Two Haemoproteus lineages, hCIRCUM03 and GAGLA03, were identical to those isolated from Corvus monedula in southern Spain and Garrulus glandarius in Bulgaria, respectively. Furthermore, the new Haemoproteus lineage hCIRCUM05 showed a 99% similarity with a lineage found infecting captive penguins in Japan., Conclusions: The comparison of the parasite lineages isolated in this study with those previously found infecting birds allowed us to identify some potential nodes in the transmission network of avian blood parasite lineages. These results highlight the complexity of the transmission networks of blood parasites in the wild that may involve a high diversity of susceptible birds and insect vectors.

Haemosporidians, a group of vector-borne parasites that include Plasmodium, infect vertebrates including birds. Although mosquitoes are crucial elements in the transmission of avian malaria parasites, little is known of their ecology as vectors. We examined the presence of Plasmodium and Haemoproteus lineages in five mosquito species belonging to the genera Culex and Ochlerotatus to test for the effect of vector species, season and host-feeding source on the transmission dynamics of these pathogens. We analyzed 166 blood-fed individually and 5,579 unfed mosquitoes (grouped in 197 pools) from a locality in southern Spain. In all, 15 Plasmodium and two Haemoproteus lineages were identified on the basis of a fragment of 478 bp of the mitochondrial cytochrome b gene. Infection prevalence of blood parasites in unfed mosquitoes varied between species (range: 0-3.2%) and seasons. The feeding source was identified in 91 mosquitoes where 78% were identified as bird. We found that i) several Plasmodium lineages are shared among different Culex species and one Plasmodium lineage is shared between Culex and Ochlerotatus genera; ii) mosquitoes harboured Haemoproteus parasites; iii) pools of unfed females of mostly ornithophilic Culex species had a higher Plasmodium prevalence than the only mammophylic Culex species studied. However, the mammophylic Ochlerotatus caspius had in pool samples the greatest Plasmodium prevalence. This relative high prevalence may be determined by inter-specific differences in vector survival, susceptibility to infection but also the possibility that this species feeds on birds more frequently than previously thought. Finally, iv) infection rate of mosquitoes varies between seasons and reaches its maximum prevalence during autumn and minimum prevalence in spring.

Earlier migration in males than in females is the commonest pattern in migrating passerines and is positively related to size dimorphism and dichromatism. The early arrival of males is a costly trait that may confer reproductive advantages in terms of better territories and/or mates. Given the physiological cost of migration, early migrants are those in best condition and accordingly the prevalence, load, and/or diversity of parasites is expected to increase in both sexes for late migrants. To test this hypothesis, we sampled 187 trans-Saharan migrant garden warblers Sylvia borin and 64 resident serins Serinus serinus (as a control for potential circannual patterns in parasite load) during spring migration in Spain. We assessed the prevalence of blood parasites (Haemoproteus, Plasmodium, and Leucocytozoon) and the prevalence and load of intestinal parasites (mainly coccidians and spirurids). The relationship between parasite (prevalence, load, and richness) and the timing of passage through a stopover area was tested using generalized linear models. Protandry occurs in the monomorphic garden warbler and males migrated on average 5.5 days before females. Intestinal parasite richness increased with the date of migration. The timing of migration was unrelated to the presence or load of the other parasite groups analyzed. Our results support the idea that the timing of migration is a condition-dependent trait and suggests that multiple intestinal parasite infestations could delay migration in birds. Even in monomorphic species parasites may play a role in sexual selection by delaying the arrival of the most infected individuals at breeding grounds, thereby further increasing the benefits of mating with early-arriving individuals.

Background: Host identification is an essential step in studies on the transmission dynamics of vector-borne disease. Nowadays, molecular tools allow the identification of vertebrate hosts to the species level. However, the proportion of successful identifications is variable and may be affected by the quality of the samples and the laboratory protocols. Here, the effect of two of these factors, namely the digestion status of mosquito blood meal and the DNA extraction procedure, on the success of host identification by amplification and sequencing of a fragment of the cytochrome oxidase 1 gene were tested., Methods: Mosquitoes collected both outdoors and indoors during 2012 in southern Spain were identified to species level and their blood meal digestion status recorded using the Sella score, a visual estimation of the digestion status of mosquito blood meals. Each mosquito was assigned randomly to one of two DNA extraction procedures: the quick and cheap HotSHOT procedure or the QIAGEN DNeasy Blood and Tissue(®) kit and their hosts identified by a molecular method., Results: Three hundred and forty-seven blood-fed mosquitoes belonging to Anopheles atroparvus (n=171), Culex perexiguus (n=84), Culex pipiens (n=43), Culex theileri (n=39), Culex modestus (n=5), Ochlerotatus caspius (n=4), Culiseta sp. (n=1) were included in this study. Overall, hosts were identified from 234 blood meals compromising at least 25 species including mammals, birds and a single reptile. The success of host identification was lower in mosquitoes with an advanced stage of blood meal digestion and for blood meals extracted using the HotSHOT procedure., Conclusions: The success of host identification decreases with the advanced stage of mosquito blood meal digestion, from 84.5% for recent blood meals to 25.0% for more digested ones. Using the QIAGEN kit, the identification success improved by 17.6%, with larger increases at more advanced stages of blood meal digestion. Availability of blood-fed females used to be very limited for studies of vector ecology, and these results may help to increase the efficiency of blood meal analyses. In addition, results obtained in this study clearly support that the potential malaria vector An. atroparvus feeds on animals located outdoors but use human-made shelters for resting after feeding.

To identify the host range of potential vectors of Dirofilaria immitis Leidy, the causal agent of canine dirofilariasis, we studied the bloodmeal origin of mosquitoes trapped on two of the Canary Islands, Gran Canaria and Tenerife, where this disease is considered hyperendemic. On Gran Canaria, mosquitoes were captured using Centers for Disease Control and Prevention (CDC) traps (outdoors) and resting in a bathroom (indoors). Only CDC traps were used to capture mosquitoes in Tenerife. The species captured in decreasing order of abundance were Culex theileri Theobald, Culex pipiens L., Culiseta longiareolata Macquart, Anopheles atroparvus van Thiel, and Anopheles cinereus Theobald. The origins of bloodmeals were identified for 121 Cx. theileri and 4 Cx. pipiens after amplification and sequencing of a fragment of the vertebrate cytochrome oxidase I (COI) gene. Cx. theileri fed on goats, sheep, dogs, cattle, cats, humans, and chickens, and Cx. pipiens fed on goats and chickens. A lower success of bloodmeal identification was obtained in mosquitoes captured resting indoors than outdoors in CDC traps, probably because of a longer time period between feeding and capture. Although most Cx. theileri fed on ruminants, this species also fed on different mammal species susceptible to dirofiliarasis, including humans, suggesting it could play a role on parasite transmission.

Surveillance, research, and control of mosquito-borne diseases such as West Nile virus require efficient methods for sampling mosquitoes. We compared the efficacy of BG-Sentinel and Centers for Disease Control and Prevention (CDC)-CO(2) traps in terms of the abundances of host-seeking and blood-fed female mosquitoes and the origin of mosquito bloodmeals. Our results indicate that BG-Sentinel traps that use CO(2) and attractants are as effective as CDC-CO(2) traps for Culex mosquito species, Ochlerotatus caspius, and they are also highly efficient at capturing Anopheles atroparvus host-seeking and blood-fed females with or without CO(2). The CDC-CO(2) trap is the least efficient method for capturing blood-fed females. BG-Sentinel traps with attractants and CO(2) were significantly better at capturing mosquitoes that had fed on mammals than the unbaited BG-Sentinel and CDC-CO(2) traps in the cases of An. atroparvus and Cx. theileri. These results may help researchers to optimize trapping methods by obtaining greater sample sizes and saving time and money.

Background: Mosquito feeding behaviour determines the degree of vector-host contact and may have a serious impact on the risk of West Nile virus (WNV) epidemics. Feeding behaviour also interacts with other biotic and abiotic factors that affect virus amplification and transmission., Methodology/principal Findings: We identified the origin of blood meals in five mosquito species from three different wetlands in SW Spain. All mosquito species analysed fed with different frequencies on birds, mammals and reptiles. Both 'mosquito species' and 'locality' explained a similar amount of variance in the occurrence of avian blood meals. However, 'season of year' was the main factor explaining the presence of human blood meals. The differences in diet resulted in a marked spatial heterogeneity in the estimated WNV transmission risk. Culex perexiguus, Cx. modestus and Cx. pipiens were the main mosquito species involved in WNV enzootic circulation since they feed mainly on birds, were abundant in a number of localities and had high vector competence. Cx. perexiguus may also be important for WNV transmission to horses, as are Cx. pipiens and Cx. theileri in transmission to humans. Estimates of the WNV transmission risk based on mosquito diet, abundance and vector competence matched the results of previous WNV monitoring programs in the area. Our sensitivity analyses suggested that mosquito diet, followed by mosquito abundance and vector competence, are all relevant factors in understanding virus amplification and transmission risk in the studied wild ecosystems. At some of the studied localities, the risk of enzootic circulation of WNV was relatively high, even if the risk of transmission to humans and horses was less., Conclusions/significance: Our results describe for first time the role of five WNV candidate vectors in SW Spain. Interspecific and local differences in mosquito diet composition has an important effect on the potential transmission risk of WNV to birds, horses and humans.

West Nile virus (WNV) is a neurotropic mosquito-transmitted flavivirus that in Eurasia, Africa, and the Americas primarily affects birds and secondarily other vertebrates. WNV has caused frequent massive episodes of wild bird mortality during its expansion throughout the Americas, and has become a regulating factor in the population dynamics of many wild bird species. On the other hand, WNV-related mortalities in wild birds have rarely been reported in the Mediterranean Basin despite its well-documented circulation, and only sporadic outbreaks in horses have been documented. The causes underlying this contrasting epidemiological pattern have never been properly described. An initial suggestion is that Mediterranean and American strains possess different pathogenicities, whereas an alternative view proposes that WNV-related disease and mortalities may have been overlooked in Europe. To test these hypotheses, between 2004 and 2006 in southern Spain we sampled tissue from 119 wild bird carcasses to detect WNV and other flaviviruses, as well as blood from 227 wild birds arriving in wildlife rehabilitation centers to test for WNV seroprevalence. No flavivirus was found in the tissue samples. The prevalence of WNV-neutralizing antibodies was 2.2%, similar to that of 800 healthy birds of the same species that were captured in the field. Our results suggest that WNV circulation during the study period did not result in any detectable effects in terms of bird morbidity or mortality.

To determine circulation of West Nile virus (WNV) during nonepidemic times, we serosurveyed wild birds of Morocco in 2008. We found antibodies against WNV in 12 (3.5%) birds, against Usutu virus in 1 (0.3%), and against both in 2 (0.6%). High WNV prevalence among juvenile birds suggests local virus circulation among resident birds.

Emerging infectious diseases represent a challenge for global economies and public health. About one fourth of the last pandemics have been originated by the spread of vector-borne pathogens. In this sense, the advent of modern molecular techniques has enhanced our capabilities to understand vector-host interactions and disease ecology. However, host identification protocols have poorly profited of international DNA barcoding initiatives and/or have focused exclusively on a limited array of vector species. Therefore, ascertaining the potential afforded by DNA barcoding tools in other vector-host systems of human and veterinary importance would represent a major advance in tracking pathogen life cycles and hosts. Here, we show the applicability of a novel and efficient molecular method for the identification of the vertebrate host's DNA contained in the midgut of blood-feeding arthropods. To this end, we designed a eukaryote-universal forward primer and a vertebrate-specific reverse primer to selectively amplify 758 base pairs (bp) of the vertebrate mitochondrial Cytochrome c Oxidase Subunit I (COI) gene. Our method was validated using both extensive sequence surveys from the public domain and Polymerase Chain Reaction (PCR) experiments carried out over specimens from different Classes of vertebrates (Mammalia, Aves, Reptilia and Amphibia) and invertebrate ectoparasites (Arachnida and Insecta). The analysis of mosquito, culicoid, phlebotomie, sucking bugs, and tick bloodmeals revealed up to 40 vertebrate hosts, including 23 avian, 16 mammalian and one reptilian species. Importantly, the inspection and analysis of direct sequencing electropherograms also assisted the resolving of mixed bloodmeals. We therefore provide a universal and high-throughput diagnostic tool for the study of the ecology of haematophagous invertebrates in relation to their vertebrate hosts. Such information is crucial to support the efficient management of initiatives aimed at reducing epidemiologic risks of arthropod vector-borne pathogens, a priority for public health.

The rapid range expansion of West Nile Virus has raised interest in understanding the population dynamics and dispersal patterns of emerging infectious diseases by wildlife. We analyzed different ecological and evolutionary factors related to West Nile Virus neutralizing antibody prevalence in 72 bird species sampled in southern Spain. Prevalence of antibodies reached its maximum during the autumn and winter in comparison to summer months. Prevalence of antibodies was directly related to body mass and migratory behaviour. The greater prevalence of antibodies observed in summer migrants can be explained, among other factors, by the diversity of localities involved in their life cycles or the geographic areas visited during their migrations. Greater prevalence in larger species was explained by their longevity because the relationship was already significant when analyzing only first year birds, and probably also involved a high attraction to vectors by larger hosts. Coloniality and winter gregarism were unrelated to the prevalence of antibodies against this highly host generalist pathogen. Evolutionary relationships between species were unrelated to differences in the prevalence of antibodies. Our results suggest larger species as good candidates for easy, faster and cheaper monitoring of local, seasonal and annual changes in WN virus serology.

The Iberian red deer (Cervus elaphus hispanicus) is an emblematic game species in Spain. To generate a battery of polymorphic markers for multiplex polymerase chain reactions for the Spanish red deer, 45 loci isolated in different species of ungulates were tested. Of the primers tested, 27 amplified but only 21 were polymorphic. Eleven of these markers were subsequently optimized for multiplex in four polymerase chain reactions. This allows analysing several molecular markers jointly to substantially reduce costs. Finally, we report descriptive summary statistics such as number of alleles for the former and also test of disequilibria and heterozygosity for the latter., (© 2008 The Authors. Journal compilation © 2008 Blackwell Publishing Ltd.)

West Nile virus (WNV) is a bird flavivirus capable of infecting horses and humans that is transmitted by blood-sucking vectors. In Europe and Africa, sporadic infections and outbreaks causing human illness and deaths have occurred and have led to 2 mutually nonexclusive hypotheses regarding the circulation of WNV in Europe: (1) the occurrence of endemic sylvatic cycles that occasionally result in human or equine infection, or (2) sporadic seeding of WNV by migratory birds from areas where the virus is endemic in Africa or elsewhere that cause local epizootic foci and eventually lead to infection in humans. To investigate these 2 possibilities, we used a micro virus-neutralization test to examine the prevalence of WNV neutralizing antibodies in 574 individuals belonging to 25 species of birds captured in spring 2004 in Seville (southern Spain). Trans-Saharan migrant species had both higher prevalences and antibody titers than resident and short-distance migrants. This result suggests that trans-Saharan migrants spend part of their life cycles in areas with greater circulation of WNV, or a closely related flavivirus, before their arrival in Spain. On the other hand, seroprevalences assessed in resident birds suggest a low level of WNV circulation in the studied locality. Aside from the question of local circulation, it thus seems that the risk for introduction of strains of WNV from Africa by migratory birds merits further field and experimental studies in Spain.

A serosurvey for neutralizing antibodies against West Nile virus (WNV) in common coots (Fulica atra) was conducted in Doñana, Spain. Antibody prevalence was highest in 2003, intermediate in 2004, and lowest in 2005. Some birds seroreverted <1 year after first capture. Seroconversion of birds suggests local circulation of the virus.

The rapid expansion of West Nile virus (WNV) throughout the New World has raised interest in understanding the population dynamics and patterns of dispersal of emerging infectious diseases by wildlife. WNV affects humans, although its main reservoirs are various species of birds. Here we analyse the prevalence of WNV-neutralizing antibodies in nearly full-grown chicks belonging to seven different species of colonial waterbirds at three localities in southern Spain. Chicks with neutralizing antibodies against WNV were detected in three species and at all three localities. However, the low antibody titres suggest the presence of antibodies is probably due to maternal transfer of antibody, presumably from exposure of the adult birds to WNV or a similar flavivirus at some stage of their lives. The analyses of the movements of tagged birds confirmed that all species with antibody visit regions that have had reports of WNV infection over the past decade.

Protozoan coccidia are one of the most common intestinal parasites in birds. Ordinary coccidian detection and quantification techniques have proved to be inaccurate for wild passerines due to the existence of marked oocyst shedding rhythms throughout the day. Previous studies have suggested that these rhythms should be taken into account when analysing coccidian load and prevalence data, but their pattern and magnitude still remain poorly known. In this study we characterised shedding rhythms in the field by means of 406 samples of faeces taken from two species of passerines with different diets: the European Serin (a granivorous species), and the Garden Warbler (an insectivorous species). Both coccidian prevalence and load were two-phased, with maximums occurring in the afternoon. Oocyst elimination remained consistently high during the second half of the day, whereas prevalence peaked during the afternoon, lowering throughout the evening. This pattern was found in both species. We found a high repeatability of prevalence and intensity when differences between the morning and afternoon were statistically controlled. As a result, we suggest that sampling periods used in the analysis of coccidian prevalence and/or load studies should take into account these differences in times of shedding and be limited to the afternoon, otherwise a statistical control of this factor will be required.

DNA sequence comparisons suggest that evolutionary rates at the rabbit IGKC1 locus can differ among allelic lineages. Here we address the question of whether population turnover rates can vary among IGKC1 alleles. We studied the distribution of sixteen IGKC1 (or b-locus) allotypes in areas comprising the aboriginal species range (Iberian peninsula). Rabbits in this area belong to one of two distantly related mitochondrial lineages (mtDNA types) A and B. In the more recent distribution area of the species, all rabbits belong to the mtDNA type B lineage, and IGKC1 alleles b4 and b5 comprise over 90% of the gene pool. These two alleles are also predominant in areas of mtDNA type B prevalence within the Iberian range. However, in areas of mtDNA type A prevalence, the b4 and b5 allotypes are rare or absent; they apparently have been replaced by serologically related, but distinct, 'endemic' variants. The cytonuclear disequilibria were highly significant, also within the subsample consisting of populations from Spain. These observations suggest that allelic persistence times for the predominant IGKC1 lineages could be shorter than the divergence time of the major mtDNA lineages A and B. In contrast, the relative gene frequencies of the IGKC1 allele b9 were similar among the type A and type B rabbits; it was present in most populations at low frequency. In consequence, persistence times of the b9 allele appear to be longer than the divergence time of lineages A and B. The data reported here are in agreement with the DNA sequence data, providing further proof that the molecular clock can run at different rates among allelic lineages at the rabbit IGKC1 locus.