Ramón C. Soriguer

Mitochondrial DNA evolution in lagomorphs: Origin of systematic heteroplasmy and organization of diversity in European rabbits

SummaryA characterization was conducted on mitochondrial DNA (mtDNA) molecules extracted separately from 107 European rabbits (Oryctolagus cuniculus) both wild and domestic, 13 European hares (Lepus capensis), and 1 eastern cottontail (Sylvilagus floridanus). Experimentally this study took into account restriction site polymorphism, overall length variation of the noncoding region, and numbers of repeated sequences. Nucleotide divergences indicate that the mtDNAs from the three species derived from a common ancestor some 6–8 million years (Myr) ago. Every animal appeared heteroplasmic for a set of molecules with various lengths of the noncoding region and variable numbers of repeated sequences that contribute to them. This systematic heteroplasmy, most probably generated by a rate of localized mtDNA rearrangements high enough to counterbalance the cellular segregation of rearranged molecules, is a shared derived character of leporids.The geographic distribution of mtDNA polymorphism among wild rabbit populations over the western European basin shows that two molecular lineages are represented, one in southern Spain, the second over northern Spain, France, and Tunisia. These two lineages derived from a common ancestor some 2 Myr ago. Their present geographical distribution may be correlated to the separation of rabbits into two stocks at the time of Mindel glaciation.Finally the distribution of mtDNA diversity exhibits a mosaic pattern both at inter- and intrapopulation levels.

Disentangling vector-borne transmission networks: a universal DNA barcoding method to identify vertebrate hosts from arthropod bloodmeals.

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 hosts 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.

Feeding Patterns of Potential West Nile Virus Vectors in South-West Spain

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.

Seroconversion in wild birds and local circulation of West Nile virus, 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.

Prevalence of West Nile virus neutralizing antibodies in colonial aquatic birds in southern Spain

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.

Prevalence of West Nile Virus Neutralizing Antibodies in Spain Is Related to the Behavior of Migratory Birds

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.

HEMATOLOGIC AND BIOCHEMICAL REFERENCE INTERVALS FOR SPANISH IBEX

We studied blood samples from 529 Spanish ibexes (Capra pyrenaica) from different Andalusian mountain ranges in southern Spain, primarily from Sierra Nevada. For each sample, 13 hematologic and 32 biochemical parameters were analyzed. Within this database, we selected values obtained from live, free-ranging, physically restrained, clinically normal animals to determine reference intervals for these parameters. Distribution of values within each parameter was determined and differences in values between sex and age classes also were determined. We found significant differences in eight biochemical parameters among male and female ibexes. Significant differences in values for 20 hematologic and biochemical parameters between age classes also were found.

Size matters: West Nile Virus neutralizing antibodies in resident and migratory birds in Spain.

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.

Host-Feeding Patterns of Native Culex pipiens and Invasive Aedes albopictus Mosquitoes (Diptera: Culicidae) in Urban Zones from Barcelona, Spain

ABSTRACT The feeding patterns of haematophagous arthropods are of major importance in the amplification and transmission of infectious disease agents to vertebrate hosts, including humans. The establishment of new vector populations in nonnative range might alter transmission networks. The Asian tiger mosquito Aedes albopictus (Skuse) represents an example of how an invasive species can alter the risk of viral transmission to humans. Blood meal molecular identification from two sympatric mosquito species (the invasive Ae. albopictus and the native Culex pipiens) was carried out by polymerase chain reaction-based methods. Samples were collected in Barcelona metropolitan area, Spain, from June to October 2009 as part of a monitoring-control program. Blood meals were identified to the species level in 30 Ae. albopictus and 43 Cx. pipiens. Ae. albopictus acquired blood exclusively from human hosts (100%), whereas Cx. pipiens fed on a diversity of avian and mammalian hosts, including 35.7% of blood meals from humans. Based on mosquito diet, our results suggest that the Ae. albopictus invasion in Spain might increase the risk of virus transmission to humans and could support local outbreaks of imported tropical viruses such as dengue and chikungunya. However, in the studied area, the presence of this invasive species would have a negligible effect on the transmission of zoonotic agents such as West Nile virus. However, Cx. pipiens could amplify and transmit West Nile virus, but avian contribution to its diet was lower than that reported in North America. Feeding patterns of these mosquito species may help to understand the flavivirus outbreaks recently reported in southwestern Europe.

Sarcoptes scabiei: Specific immune response to sarcoptic mange in the Iberian ibex Capra pyrenaica depends on previous exposure and sex.

Host acquired immunity is a critical factor that conditions the survival of parasites. Nevertheless, there is a shortage of data concerning inter-individual immunological inequalities in wild mammals. Sarcoptic mange is a widespread parasitosis that severely affects mammals such as the Iberian ibex (Capra pyrenaica). Despite some work on the subject, the immune response to sarcoptic mange infestation is still a complex and poorly understood phenomenon. To improve knowledge of the host-Sarcoptes immunological interaction, 18 Iberian ibexes were experimentally infested. IgG levels were assessed using ELISA to test for potential factors determining the specific immune response to infestation. Previous exposure and sex appeared to affect the IgG response to infestation and our results suggest a sex-biased immunomodulation. We discuss the immunological pattern of host-Sarcoptes interactions and also suggest further lines of work that may improve the understanding of immunological interactions of host-Sarcoptes systems.