Comparative performance of microscopy, nested PCR, and real-time PCR for screening avian haemosporidian parasites in Afrotropical starlings (family Sturnidae).

Prevalence studies of avian haemosporidian parasites frequently use microscopy and the nested polymerase chain reaction (PCR) protocols for detecting infections. Newer PCR protocols to detect parasites are being developed, with the distinct advantage of reducing screening cost and time, as well as increasing efficiency and sensitivity. The detection ability of microscopy and nested PCR was compared against a real-time PCR (qPCR) protocol using genomic DNA extracted from 240 bird blood samples collected from three starling species (Cape Starling, the Greater Blue-eared Starling, and the Wattled Starling; family Sturnidae) in the Kruger national park, South Africa. All three protocols successfully detected avian haemosporidian parasites with the qPCR having a considerable edge against the other two methods. Fifteen unique cytochrome b lineages were identified of which seven were new lineages. Microscopy and nested PCR recorded similar prevalence (32.92% and 35.42% respectively). The qPCR protocol used here, although more sensitive (52.92% prevalence), is not able to differentiate between parasite genera but provides the opportunity to screen a large number of samples in large-scale studies within a specific region. This study recommends the development and adoption of new molecular protocols with increased sensitivity and accuracy in prevalence studies. Nevertheless, microscopy remains essential for the morphological description of parasites and for distinguishing between abortive and successful chronic infections. The PCR-based method displays the detection of the parasitic genome but does not reveal whether parasites have or will develop into a successful infection.

Habitat fragmentation matters more than habitat loss: The case of host-parasite interactions.

While ecologists agree that habitat loss has a substantial negative effect on biodiversity it is still very much a matter of debate whether habitat fragmentation has a lesser effect and whether this effect is positive or negative for biodiversity. Here, we assess the relative influence of tropical forest loss and fragmentation on the prevalence of vector-borne blood parasites of the genera Plasmodium and Haemoproteus in six forest bird species. We also determine whether habitat loss and fragmentation are associated with a rise or fall in prevalence. We sample more than 4000 individual birds from 58 forest sites in Guadeloupe and Martinique. Considering 34 host-parasite combinations independently and a fine characterization of the amount and spatial configuration of habitat, we use partial least square regressions to disentangle the relative effects of forest loss, forest fragmentation, landscape heterogeneity, and local weather conditions on spatial variability of parasite prevalence. Then we test for the magnitude and the sign of the effect of each environmental descriptor. Strikingly, we show that forest fragmentation explains twice as much of the variance in prevalence as habitat loss or landscape heterogeneity. In addition, habitat fragmentation leads to an overall rise in prevalence in Guadeloupe, but its effect is variable in Martinique. Both habitat loss and landscape heterogeneity exhibit taxon-specific effects. Our results suggest that habitat loss and fragmentation may have contrasting effects between tropical and temperate regions and that inter-specific interactions may not respond in the same way as more commonly used biodiversity metrics such as abundance and diversity.

Avian haemosporidia in native and invasive sparrows at an Afrotropical region.

Bio-invasions are a major threat to biodiversity and ecosystems globally and may contribute to the proliferation of emerging infectious diseases. We examined the prevalence and phylogenetic diversity of avian haemosporidian parasites infecting the non-native house sparrows (Passer domesticus) and the native southern grey-headed sparrows (Passer diffusus). Blood samples from 104 sparrows (74 house sparrows and 30 southern grey-headed sparrows) mist-netted inside and around the Kruger National Park were used. Genomic DNA was extracted from each blood sample and subjected to nested PCR analyses, Sanger sequencing and phylogenetic analyses. Overall, 35.57% (37/104) of the birds sampled were infected with at least one haemosporidian parasites. Southern grey-headed sparrows had a higher parasite prevalence (60%) than house sparrows (24.3%). A total of 16 parasite lineages were identified, of which eight were novel lineages. Whereas Haemoproteus spp. showed the highest lineage diversity, Leucocytozoon spp. were the most prevalent parasites, albeit with significant differences between sparrow species. A single Plasmodium sp. infection was recorded in a southern grey-headed sparrow. In support of the enemy release hypothesis, we found that prevalence on non-native house sparrows was lower than prevalence recorded in their region of origin and also that they were infected only by indigenous parasites lineages.

Water for African elephants (Loxodonta africana): faecal microbial loads affect use of artificial waterholes.

In semi-arid protected areas, artificial waterholes ensure that water is locally available to animals for extended periods. However, artificial waterholes may limit animal movement, which contributes towards habitat deterioration. Challenges of artificial water provisioning worsen in the presence of ecosystem engineers like African elephants Loxodonta africana, capable of transforming environments. Camera traps were used to monitor elephant visitation at 21 artificial waterholes in the Kruger National Park, South Africa. We also assessed if water quality parameters influenced elephant preference for certain waterholes. There were no significant correlations between elephant abundance and water physicochemical properties. However, there was a strong negative correlation between elephant abundance and levels of Escherichia coli in water. Our findings suggest that elephants avoid drinking water with high levels of faecal microbial loads. Whereas most studies addressing animal management in protected areas consider waterholes as homogeneous units, we posit that water quality could also determine local landscape use and movement patterns of key species like elephants, a finding with relevant implications in reserve management practices.

High diversity and low genetic structure of feather mites associated with a phenotypically variable bird host.

Obligate symbionts may be genetically structured among host individuals and among phenotypically distinct host populations. Such processes may in turn determine within-host genetic diversity of symbionts, which is relevant for understanding symbiont population dynamics. We analysed the population genetic structure of two species of feather mites (Proctophyllodes sylviae and Trouessartia bifurcata) in migratory and resident blackcaps Sylvia atricapilla that winter sympatrically. Resident and migratory hosts may provide mites with habitats of different qualities, what might promote specialization of mite populations. We found high genetic diversity of within-host populations for both mite species, but no sign of genetic structure of mites between migratory and resident hosts. Our results suggest that, although dispersal mechanisms between hosts during the non-breeding season are unclear, mite populations are not limited by transmission bottlenecks that would reduce genetic diversity among individuals that share a host. Additionally, there is no evidence that host phenotypic divergence (associated with the evolution of migration and residency) has promoted the evolution of host-specialist mite populations. Unrestricted dispersal among host types may allow symbiotic organisms to avoid inbreeding and to persist in the face of habitat heterogeneity in phenotypically diverse host populations.

Blood parasites in hooded crows (Corvus corone cornix) in Northwest Italy.

Haemoparasites and their effects on hooded crows (Corvus corone cornix) are poorly studied. The aims are to evaluate the prevalence of Haemoproteus spp./Plasmodium spp. or Leucocytozoon spp., to correlate this with gross and histopathological findings, and to investigate the association among infection and geographical origin, age, gender, parasite distribution and prevalence among organs. Hooded crows (n = 47) were collected within a regional culling programme from 3 districts in the province of Turin (Italy) and subjected to necropsy. Histological and molecular analyses were carried out on some tissues. Leucocytozoon spp. was detected in 46 crows (97.9%) by polymerase chain reaction (PCR), whereas 28 birds (59.6%) were found to be positive for Haemoproteus spp./Plasmodium spp. The distribution of parasites in several organs varied significantly, showing that Leucocytozoon spp. is ubiquitous in organs in contrast with Haemoproteus spp./Plasmodium spp., which have a specific predilection for spleen and lungs. The prevalence of Haemoproteus spp./Plasmodium spp. also differed significantly among the crows captured in the areas of the study. The high prevalence of haemoparasites emphasizes the success of ornithophilic vectors and the susceptibility of this species to infection. Differences in prevalence among the sites are probably due to orographic features of the areas, variations in vector species and density, or to crow population size or structure. In spite of the high infection rate, no gross and histological lesions were found. This finding further suggests an evolutionary adaptation between crows and avian blood parasites.

Evolution of seasonal transmission patterns in avian blood-borne parasites.

In temperate regions, many vector-borne parasites maximise their transmission prospects by adjusting reproduction to seasonal cycles of host susceptibility and vector availability. Nevertheless, in these regions there are areas where environmental conditions are favourable throughout the year, so that parasites could benefit from a year-round transmission strategy. We analysed how different transmission strategies (strict summer transmission, extended summer transmission - including spring and autumn, and year round transmission) have evolved among the different genetic lineages of Haemoproteus parabelopolskyi, an avian blood-borne parasite shared by three sibling species of passerine hosts. Our results indicate that the ancestral state of this clade of parasites had a strict summer transmission with the blackcap (Sylvia atricapilla) as the host. Other transmission strategies and switches to the other host species (Sylvia abyssinica and Sylvia borin) evolved recently, several times, independently. This suggests that, although year-round transmission is ecologically successful at present, seasonal transmission may have become more stable over evolutionary time. Switches from strict summer to an extended or year-round transmission strategy could have ecological consequences, if they promote the spread of parasites into more distant regions, transported by the migrating bird hosts. Therefore, a deeper knowledge of how different parasite transmission strategies are structured among birds in temperate areas is essential for understanding how disease emergence risks may develop in the future.

Prevalence and diversity of Plasmodium and Haemoproteus parasites in the globally-threatened Aquatic Warbler Acrocephalus paludicola.

The diversity and prevalence of malaria parasites of the genera Plasmodium and Haemoproteus were determined in the globally-threatened Aquatic Warbler Acrocephalus paludicola. Birds were sampled during migration in Portugal and at the wintering quarters in Senegal and parasites were detected using molecular methods. Only three generalist parasite lineages (Plasmodium) were found. There were no significant differences in the prevalence of parasites between sexes in Europe, but adults had higher prevalence than first-year birds, and birds in Europe had higher prevalence than those captured in Africa. When comparing with other Acrocephalus species and taking sample size into account, Aquatic Warblers had the lowest prevalence and, together with another threatened species, the Seychelles Warbler Acrocephalus sechellensis, the lowest diversity of malaria parasites. We hypothesize that the low diversity of parasites and absence of specialist lineages of Aquatic Warblers are caused by its small population size and fragmented distribution. Furthermore, Aquatic Warblers' extreme habitat specialization may decrease their exposure to malaria parasites, but other explanations such as high mortality (which would constraint the sampling of infected birds) or, in contrast, very efficient immunological system in clearing the infections cannot be ruled out. This study contributes to explain variation in prevalence and diversity of malaria parasites among hosts.

Different space preferences and within-host competition promote niche partitioning between symbiotic feather mite species.

Obligate symbionts (including parasites, commensals and mutualists) often share host species and host-based food resources. Such symbionts are frequently distributed unequally among hosts with different phenotypic features, or occupy different regions on a host. However, the processes leading to distinct within-host symbiont distributions remain obscure. We aimed to test whether distinct in-host symbiont distributions arise as the outcome of species-specific habitat preferences or interspecific competition, and how host phenotype influences such processes. To this end, we studied the distribution within and among individual bird hosts of two feather mites (Proctophyllodes sylviae and Trouessartia bifurcata) of migratory and sedentary European blackcaps, Sylvia atricapilla, wintering in sympatry. Trouessartia bifurcata was mostly restricted to resident blackcaps, while P. sylviae was abundant on both host types. Within hosts, each species tended to settle on different feather sectors (proximal or distal, respectively), which they filled by spreading on the wing following ordered but opposite patterns, thereby supporting the view that spatial segregation was primarily the outcome of dissimilar space preferences. However, we also found evidence of competition finely tuning mite distributions: when P. sylviae increased abundance and expanded onto the range of T. bifurcata, abundances of the two species were negatively correlated in the shared areas. In addition, the presence of T. bifurcata on a host was associated with a more restricted distribution of P. sylviae. Our results show that both species-specific preferences and interspecific interactions contribute to shaping mite distributions among and on individual hosts, a situation likely mirrored by other host-multi-symbiont systems.

Prevalence of new and known species of haemoparasites in feral pigeons in northwest Italy.

BACKGROUND: Haemoparasites in feral pigeons have been studied in several countries but no data are available from Italy. The aim of this work was to evaluate the prevalence and diversity of Haemoproteus spp./Plasmodium spp. and Leucocytozoon spp. in feral pigeons from northwest Italy, as well as the association between infection and host age or sex. METHODS: Feral pigeons were collected during a regional culling programme from the Piedmont region (northwest Italy) and subjected to necropsy. Infections were detected from DNA extracted from the spleen following a nested PCR protocol. The association between sex or age and infection status was evaluated using the chi-squared test for independence or Fisher's exact test. RESULTS: Out of 51 animals, 15 were positive for Haemoproteus/Plasmodium spp. and eight for Leucocytozoon spp., with a significant difference between haemoparasites prevalence. There was no significant association between age or sex and infection status. The coinfection with different haemoparasites was very significant (p < 0.01), showing a greater relative risk to be infected by a second haemoparasite in birds already infected, in particular in male and in adult pigeons. DNA sequencing of Leucocytozoon spp. showed six different lineages in pigeons, and one of Haemoproteus and Plasmodium, respectively. CONCLUSIONS: Blood parasites are continuously circulating around the world, and the results presented in the paper suggest that cross infection of feral pigeons with haemoparasites typical of other migratory or nonmigratory bird species is possible. Moreover, the geographical location of Italy along the main migratory routes is a crucial factor to be considered for migratory birds, because they can be affected by blood parasites detected in feral pigeons, and vice versa.

Global warming will reshuffle the areas of high prevalence and richness of three genera of avian blood parasites.

The importance of parasitism for host populations depends on local parasite richness and prevalence: usually host individuals face higher infection risk in areas where parasites are most diverse, and host dispersal to or from these areas may have fitness consequences. Knowing how parasites are and will be distributed in space and time (in a context of global change) is thus crucial from both an ecological and a biological conservation perspective. Nevertheless, most research articles focus just on elaborating models of parasite distribution instead of parasite diversity. We produced distribution models of the areas where haemosporidian parasites are currently highly diverse (both at community and at within-host levels) and prevalent among Iberian populations of a model passerine host: the blackcap Sylvia atricapilla; and how these areas are expected to vary according to three scenarios of climate change. On the basis of these models, we analysed whether variation among populations in parasite richness or prevalence are expected to remain the same or change in the future, thereby reshuffling the geographic mosaic of host-parasite interactions as we observe it today. Our models predict a rearrangement of areas of high prevalence and richness of parasites in the future, with Haemoproteus and Leucocytozoon parasites (today the most diverse genera in blackcaps) losing areas of high diversity and Plasmodium parasites (the most virulent ones) gaining them. Likewise, the prevalence of multiple infections and parasite infracommunity richness would be reduced. Importantly, differences among populations in the prevalence and richness of parasites are expected to decrease in the future, creating a more homogeneous parasitic landscape. This predicts an altered geographic mosaic of host-parasite relationships, which will modify the interaction arena in which parasite virulence evolves.

Finding the appropriate variables to model the distribution of vector-borne parasites with different environmental preferences: climate is not enough.

Understanding how environmental variation influences the distribution of parasite diversity is critical if we are to anticipate disease emergence risks associated with global change. However, choosing the relevant variables for modelling current and future parasite distributions may be difficult: candidate predictors are many, and they seldom are statistically independent. This problem often leads to simplistic models of current and projected future parasite distributions, with climatic variables prioritized over potentially important landscape features or host population attributes. We studied avian blood parasites of the genera Plasmodium, Haemoproteus and Leucocytozoon (which are viewed as potential emergent pathogens) in 37 Iberian blackcap Sylvia atricapilla populations. We used Partial Least Squares regression to assess the relative importance of a wide array of putative determinants of variation in the diversity of these parasites, including climate, landscape features and host population migration. Both prevalence and richness of parasites were predominantly related to climate (an effect which was primarily, but not exclusively driven by variation in temperature), but landscape features and host migration also explained variation in parasite diversity. Remarkably, different models emerged for each parasite genus, although all parasites were studied in the same host species. Our results show that parasite distribution models, which are usually based on climatic variables alone, improve by including other types of predictors. Moreover, closely related parasites may show different relationships to the same environmental influences (both in magnitude and direction). Thus, a model used to develop one parasite distribution can probably not be applied identically even to the most similar host-parasite systems.

Molecular characterization of haemosporidian parasites from kites of the genus Milvus (Aves: Accipitridae).

Despite the ecological significance and appeal of birds of prey, many aspects of their biology remain poorly known, including the diversity of parasites infecting them in the wild. We studied the diversity and prevalence of haemosporidian parasites infecting the two species of kites of the genus Milvus, aiming to describe the phylogenetic relationships among them and with other haemosporidians, as well as their distribution in the two host species. Black kites, Milvus migrans, harboured a more diverse community of parasites, including three haplotypes of each of the three genera Plasmodium, Haemoproteus and Leucocytozoon, which also occurred at a higher prevalence than in red kites. In red kites, Milvus milvus only three haplotypes of Leucocytozoon were found. Kite parasites were not closely related to one another nor were they kite-specific: their diversity spanned various branches of the haemosporidian phylogenetic tree, and their closest relatives were found in other species (including various avian orders), although some Leucocytozoon and Haemoproteus haplotypes clustered within apparently raptor-specific parasite clades. Remarkably, Plasmodium spp. and Haemoproteus spp. infected adult black kites only, an observation which supports the hypothesis that they are transmitted at the African wintering grounds, while Leucocytozoon spp. is putatively transmitted only in Europe. Intercontinental migration of the black kite might explain the divergence of parasite diversity between these two sister species.