Avian Malaria Lineages Research Articles

Molecular characterization of avian malaria in the spotless starling (Sturnus unicolor).

We studied the prevalence and genetic diversity of malaria parasites in the poorly investigated spotless starling (Sturnus unicolor) breeding in central Spain, aiming to describe the phylogenetic relationships among them and with other haemosporidians infecting the genus Sturnus. A total of 180 nestlings and 180 adult individuals from four different breeding seasons were screened for haemosporidian parasites using a nested PCR approach for the genera Plasmodium and Haemoproteus. Although the malaria prevalence ranged between years, the overall prevalence was 6.94%. Adults had a higher prevalence than chicks: 12.77 vs. 1.11%, respectively. We molecularly characterized avian malaria isolated in peripheral blood samples taken from malaria-infected individuals. Sequence analyses revealed four unique Plasmodium lineages of avian malaria (STURUNI01, STURUNI02, SYAT05, SGS1) in our spotless starling population. The phylogenetic analysis showed a well-supported clade comprised by STURUNI01, STURUNI02, and SYAT05. The most common lineage (SYAT05) has been previously found in 26 other avian host species, including populations of spotless starling in Portugal. Because this sedentary species is widely distributed throughout the Iberian Peninsula, we suggest that the local transmission of these lineages might place migratory birds at infection risk.

Development of a rapid HRM qPCR for the diagnosis of the four most prevalent Plasmodium lineages in New Zealand.

Although wildlife rehabilitation and translocations are important tools in wildlife conservation in New Zealand, disease screening of birds has not been standardized. Additionally, the results of the screening programmes are often difficult to interpret due to missing disease data in resident or translocating avian populations. Molecular methods have become the most widespread method for diagnosing avian malaria (Plasmodium spp.) infections. However, these methods can be time-consuming, expensive and are less specific in diagnosing mixed infections. Thus, this study developed a new real-time PCR (qPCR) method that was able to detect and specifically identify infections of the three most common lineages of avian malaria in New Zealand (Plasmodium (Novyella) sp. SYAT05, Plasmodium elongatum GRW6 and Plasmodium spp. LINN1) as well as a less common, pathogenic Plasmodium relictum GRW4 lineage. The assay was also able to discern combinations of these parasites in the same sample and had a detection limit of five parasites per microlitre. Due to concerns relating to the presence of the potentially highly pathogenic P. relictum GRW4 lineage in avian populations, an additional confirmatory high resolution (HRM) qPCR was developed to distinguish between commonly identified P. elongatum GRW6 from P. relictum GRW4. The new qPCR assays were tested using tissue samples containing Plasmodium schizonts from three naturally infected dead birds resulting in the identified infection of P. elongatum GRW6. Thus, these rapid qPCR assays have shown to be cost-effective and rapid screening tools for the detection of Plasmodium infection in New Zealand native birds.

Co-infections and environmental conditions drive the distributions of blood parasites in wild birds.

Experimental work increasingly suggests that non-random pathogen associations can affect the spread or severity of disease. Yet due to difficulties distinguishing and interpreting co-infections, evidence for the presence and directionality of pathogen co-occurrences in wildlife is rudimentary. We provide empirical evidence for pathogen co-occurrences by analysing infection matrices for avian malaria (Haemoproteus and Plasmodium spp.) and parasitic filarial nematodes (microfilariae) in wild birds (New Caledonian Zosterops spp.). Using visual and genus-specific molecular parasite screening, we identified high levels of co-infections that would have been missed using PCR alone. Avian malaria lineages were assigned to species level using morphological descriptions. We estimated parasite co-occurrence probabilities, while accounting for environmental predictors, in a hierarchical multivariate logistic regression. Co-infections occurred in 36% of infected birds. We identified both positively and negatively correlated parasite co-occurrence probabilities when accounting for host, habitat and island effects. Two of three pairwise avian malaria co-occurrences were strongly negative, despite each malaria parasite occurring across all islands and habitats. Birds with microfilariae had elevated heterophil to lymphocyte ratios and were all co-infected with avian malaria, consistent with evidence that host immune modulation by parasitic nematodes facilitates malaria co-infections. Importantly, co-occurrence patterns with microfilariae varied in direction among avian malaria species; two malaria parasites correlated positively but a third correlated negatively with microfilariae. We show that wildlife co-infections are frequent, possibly affecting infection rates through competition or facilitation. We argue that combining multiple diagnostic screening methods with multivariate logistic regression offers a platform to disentangle impacts of environmental factors and parasite co-occurrences on wildlife disease.

A quantitative PCR protocol for detecting specific Haemoproteus lineages: molecular characterization of blood parasites in a Sedge Warbler population from southern Poland

AbstractIn evolutionary studies, blood parasites in avian populations are commonly used as a model of host–parasite interactions. The effect of mixed infections on avian hosts has recently drawn more interest, but the effects of infection with multiple blood parasites and specific parasite lineages are poorly known. A protocol for reliable detection and quantification of lineages is essential to this type of research. Here, we present a newly developed quantitative PCR (qPCR) assay using SYBR Green I to assess the infection intensity of SW1 and SW3 Haemoproteus belopolskyi lineages in a Sedge Warbler (Acrocephalus schoenobaenus) population from southern Poland. These Haemoproteus lineages are not specific to the Sedge Warbler, so the proposed protocol should prove useful for many avian malaria studies. The assay is based on nucleotide primers designed to amplify a fragment of the cytochrome b gene, allowing the two avian malaria lineages to be differentiated. Using this assay, specific host–parasite interactions can be identified and the impact of mixed infections on a host population can be assessed. Most of the parasitized birds in our study were in a low-intensity, chronic phase of infection. In those with mixed SW1/SW3 infections, we detected significantly higher parasitemia caused by the SW3 lineage, whose prevalence was underestimated by the commonly used method, nested PCR. The prevalence of avian malaria parasites in the studied population as estimated by nested PCR was 61 % and did not differ between years, though the prevalence of the SW1 lineage showed significant annual variation. Altogether, two Haemoproteus and five Plasmodium lineages were detected. The two Haemoproteus lineages (SW1, SW3) were most prevalent in the population and comprised 93 % of all infections. We detected significantly higher haemoparasite prevalence and intensity in males, which were sampled immediately after arrival from wintering grounds, suggesting decreased immunoprotection as a result of adaptive resource allocation during migration.

Temporal changes in mosquito abundance (Culex pipiens), avian malaria prevalence and lineage composition.

BackgroundKnowledge on the temporal dynamics of host/vector/parasite interactions is a pre-requisite to further address relevant questions in the fields of epidemiology and evolutionary ecology of infectious diseases. In studies of avian malaria, the natural history of Plasmodium parasites with their natural mosquito vectors, however, is mostly unknown.MethodsUsing artificial water containers placed in the field, we monitored the relative abundance of parous females of Culex pipiens mosquitoes during two years (2010–2011), in a population in western Switzerland. Additionally, we used molecular tools to examine changes in avian malaria prevalence and Plasmodium lineage composition in female C. pipiens caught throughout one field season (April-August) in 2011.ResultsC. pipiens relative abundance varied both between years and months, and was associated with temperature fluctuations. Total Plasmodium prevalence was high and increased from spring to summer months (13.1-20.3%). The Plasmodium community was composed of seven different lineages including P. relictum (SGS1, GRW11 and PADOM02 lineages), P. vaughani (lineage SYAT05) and other Plasmodium spp. (AFTRU5, PADOM1, COLL1). The most prevalent lineages, P. vaughani (lineage SYAT05) and P. relictum (lineage SGS1), were consistently found between years, although they had antagonistic dominance patterns during the season survey.ConclusionsOur results suggest that the time window of analysis is critical in evaluating changes in the community of avian malaria lineages infecting mosquitoes. The potential determinants of the observed changes as well as their implications for future prospects on avian malaria are discussed.

MHC-I Affects Infection Intensity but Not Infection Status with a Frequent Avian Malaria Parasite in Blue Tits

Host resistance against parasites depends on three aspects: the ability to prevent, control and clear infections. In vertebrates the immune system consists of innate and adaptive immunity. Innate immunity is particularly important for preventing infection and eradicating established infections at an early stage while adaptive immunity is slow, but powerful, and essential for controlling infection intensities and eventually clearing infections. Major Histocompatibility Complex (MHC) molecules are central in adaptive immunity, and studies on parasite resistance and MHC in wild animals have found effects on both infection intensity (parasite load) and infection status (infected or not). It seems MHC can affect both the ability to control infection intensities and the ability to clear infections. However, these two aspects have rarely been considered simultaneously, and their relative importance in natural populations is therefore unclear. Here we investigate if MHC class I genotype affects infection intensity and infection status with a frequent avian malaria infection Haemoproteus majoris in a natural population of blue tits Cyanistes caeruleus. We found a significant negative association between a single MHC allele and infection intensity but no association with infection status. Blue tits that carry a specific MHC allele seem able to suppress H. majoris infection intensity, while we have no evidence that this allele also has an effect on clearance of the H. majoris infection, a result that is in contrast with some previous studies of MHC and avian malaria. A likely explanation could be that the clearance rate of avian malaria parasites differs between avian malaria lineages and/or between avian hosts.

Next-generation sequencing reveals cryptic mtDNA diversity of Plasmodium relictum in the Hawaiian Islands

Next-generation 454 sequencing techniques were used to re-examine diversity of mitochondrial cytochrome b lineages of avian malaria (Plasmodium relictum) in Hawaii. We document a minimum of 23 variant lineages of the parasite based on single nucleotide transitional changes, in addition to the previously reported single lineage (GRW4). A new, publicly available portal (Integroomer) was developed for initial parsing of 454 datasets. Mean variant prevalence and frequency was higher in low elevation Hawaii Amakihi (Hemignathus virens) with Avipoxvirus-like lesions (P = 0·001), suggesting that the variants may be biologically distinct. By contrast, variant prevalence and frequency did not differ significantly among mid-elevation Apapane (Himatione sanguinea) with or without lesions (P = 0·691). The low frequency and the lack of detection of variants independent of GRW4 suggest that multiple independent introductions of P. relictum to Hawaii are unlikely. Multiple variants may have been introduced in heteroplasmy with GRW4 or exist within the tandem repeat structure of the mitochondrial genome. The discovery of multiple mitochondrial lineages of P. relictum in Hawaii provides a measure of genetic diversity within a geographically isolated population of this parasite and suggests the origins and evolution of parasite diversity may be more complicated than previously recognized.

Plasmodium relictum (lineages pSGS1 and pGRW11): Complete synchronous sporogony in mosquitoes Culex pipiens pipiens

Plasmodium relictum is a widespread invasive agent of avian malaria, responsible for acute, chronic and debilitating diseases in many species of birds. Recent PCR-based studies revealed astonishing genetic diversity of avian malaria parasites (genus Plasmodium), with numerous genetic lineages deposited in GenBank. Many studies addressed distribution and evolutionary relationships of avian Plasmodium lineages, but information about patterns of development of different lineages in mosquito vectors remains insufficient. Here we present data on sporogonic development of 2 widespread mitochondrial cytochrome b lineages (cyt b) of P. relictum (pSGS1 and pGRW11) in mosquito Culex pipiens pipiens. Genetic distance between these lineages is 0.2%; they fall in a well-supported clade in the phylogenetic tree. Three P. relictum strains were isolated from common crossbill (Loxia curvirostra, lineage pSGS1), domestic canary (Serinus canaria domestica, pSGS1) and house sparrow (Passer domesticus, pGRW11). These strains were multiplied in domestic canaries and used as donors of malarial gametocytes to infect C. p. pipiens. Mosquitoes were allowed to take blood meal on infected canaries and then dissected on intervals to study development of sporogonic stages. All 3 strains developed synchronously and completed sporogony in this vector, with infective sporozoites reported in the salivary glands on the day 14 after infection. Ookinetes, oocysts and sporozoites of all strains were indistinguishable morphologically. This study shows that patterns of sporogonic development of the closely related lineages pSGS1 and pGRW11 and different strains of the lineage pSGS1 of P. relictum are similar indicating that phylogenetic trees based on the cyt b gene likely can be used for predicting sporogonic development of genetically similar avian malaria lineages in mosquito vectors.

PRESENCE AND SEASONAL PREVALENCE OF PLASMODIUM SPP. IN A RARE ENDEMIC NEW ZEALAND PASSERINE (TIEKE OR SADDLEBACK, PHILESTURNUS CARUNCULATUS)

The conservation and management of Saddlebacks (Philesturnus carunculatus) and other New Zealand birds, currently relies on the translocation of individuals to predator-free sites. Avian malaria has been identified as one of the diseases to be tested for prior to translocations in New Zealand, with the aim of translocating disease-free individuals. We describe avian malaria lineages and their seasonal prevalence in 2007-2008 in Saddlebacks from Mokoia Island, a source of birds for translocations, and investigate their pathogenicity. Three lineages of avian malaria were found at low prevalence (≤10.6%) and parasitemia (all but one infection were below 1/10,000 erythrocytes), typical of chronic infections. Two lineages clustered with previously identified lineages of Plasmodium relictum and one with a lineage of Plasmodium (Huffia) elongatum. Prevalence of malaria infection was higher in the spring with no significant difference in prevalence between juvenile and adult birds. We found no effect of stress on infections or any indication of pathogenicity.

Prevalence and diversity patterns of avian blood parasites in degraded African rainforest habitats

Land use changes including deforestation, road construction and agricultural encroachments have been linked to the increased prevalence of several infectious diseases. In order to better understand how deforestation affects the prevalence of vector-borne infectious diseases in wildlife, nine paired sites were sampled (disturbed vs. undisturbed habitats) in Southern Cameroon. We studied the diversity, prevalence and distribution of avian malaria parasites (Plasmodium spp.) and other related haemosporidians (species of Haemoproteus and Leucocytozoon) from these sites in two widespread species of African rainforest birds, the yellow-whiskered greenbul (Andropadus latirostris, Pycnonotidae) and the olive sunbird (Cyanomitra olivacea, Nectariniidae). Twenty-six mitochondrial cytochrome b lineages were identified: 20 Plasmodium lineages and 6 Haemoproteus lineages. These lineages showed no geographic specificity, nor significant differences in lineage diversity between habitat types. However, we found that the prevalence of Leucocytozoon and Haemoproteus infections were significantly higher in undisturbed than in deforested habitats (Leucocytozoon spp. 50.3% vs. 35.8%, Haemoproteus spp. 16.3% vs. 10.8%). We also found higher prevalence for all haemosporidian parasites in C. olivacea than in A. latirostris species (70.2% vs. 58.2%). Interestingly, we found one morphospecies of Plasmodium in C. olivacea, as represented by a clade of related lineages, showed increased prevalence at disturbed sites, while another showed a decrease, testifying to different patterns of transmission, even among closely related lineages of avian malaria, in relation to deforestation. Our work demonstrates that anthropogenic habitat change can affect host-parasite systems and result in opposing trends in prevalence of haemosporidian parasites in wild bird populations.

Genetic characterization, distribution and prevalence of avian pox and avian malaria in the Berthelot’s pipit (Anthus berthelotii) in Macaronesia

Exotic pathogens have been implicated in the decline and extinction of various native-island-bird species. Despite the fact that there is increasing concern about the introduction of diseases in island ecosystems, little is known about parasites in the islands of Macaronesia. We focus on Berthelot's pipit (Anthus berthelotii), an endemic and widespread Macaronesian bird species, using a combination of field studies and molecular techniques to determine: (1) the range and prevalence of avian pox and malaria in Berthelot's pipits throughout the species' distribution, (2) the genetic characterization of both parasites in order to ascertain the level of host specificity. We sampled 447 pipits across the 12 islands inhabited by this species. Overall, 8% of all individuals showed evidence of pox lesions and 16% were infected with avian malaria, respectively. We observed marked differences in the prevalence of parasites among islands both within and between archipelagos. Avian pox prevalence varied between 0-54% within and between archipelagos and avian malaria prevalence varied between 0-64% within and between archipelagos. The diversity of pathogens detected was low: only two genetic lineages of avian malaria and one lineage of avian pox were found to infect the pipit throughout its range. Interestingly, both avian malaria parasites found were Plasmodium spp. that had not been previously reported in the Macaronesian avifauna (but that had been observed in the lesser kestrel Falco naumannii), while the avian pox was a host specific lineage that had previously been reported on two of the Canary Islands.

Consequences of chronic infections with three different avian malaria lineages on reproductive performance of Lesser Kestrels (Falco naumanni)

We studied the consequences of chronic infections by three different lineages of avian malaria, two Plasmodium (RTSR1, LK6) and one Haemoproteus (LK2), on reproductive performance of Lesser Kestrels (Falco naumanni). Malaria infections in male and female parents had no effect on clutch size, hatching success or nesting success. However, when only successful nests were considered, we found that males parasitized by LK6 raised a lower number of fledglings, suggesting that the level of parental effort by males may be limited by this particular lineage of Plasmodium. This effect was not evident in females, probably due to the higher investment of males during the chick rearing period in this species. Overall, we have found that chronic stages of specific malaria lineages have certain negative consequences on host reproductive performance, highlighting the importance of considering genetic differences among malaria parasites to study their consequences on natural bird populations.

No relationship between individual genetic diversity and prevalence of avian malaria in a migratory kestrel

Insight into the genetic basis of malaria resistance is crucial for understanding the consequences of this parasite group on animal populations. Here, we analyse the relationship between genotypic variation at 11 highly variable microsatellite loci and prevalence of three different lineages of avian malaria, two Plasmodium (RTSR1, LK6) and one Haemoproteus (LK2), in a wild population of the endangered lesser kestrel (Falco naumanni). Although we used a large sample size (584 typed individuals), we did not find any significant association between the prevalence of the studied parasite lineages and individual genetic diversity. Although our data set is large, the 11 neutral markers typed may have had low power to detect such association, in part because of the low parasite prevalence observed (less than 5% of infected birds). However, the fact that we have detected previous correlations between genetic diversity and other traits (ectoparasitism risk, fecundity) in the study population using the same panel of neutral markers and lower sample sizes suggests that other factors could underlie the absence of such a similar correlation with avian malaria. Differences in the genetics of the studied traits and in their particular basis of inbreeding depression (dominance vs. overdominance) may have led to malaria prevalence, but not other traits, being uncoupled with individual genetic diversity. Also, we cannot discard the possibility that the absence of association was a consequence of a low pathogenic effect of these particular malaria lineages on our lesser kestrel population, and thus we should not expect the evolution of genetic resistance against these parasites.

Within‐population variation in prevalence and lineage distribution of avian malaria in blue tits, Cyanistes caeruleus

The development of molecular genetic screening techniques for avian blood parasites has revealed many novel aspects of their ecology, including greatly elevated diversity and complex host-parasite relationships. Many previous studies of malaria in birds have treated single study populations as spatially homogeneous with respect to the likelihood of transmission of malaria to hosts, and we have very little idea whether any spatial heterogeneity influences different malaria lineages similarly. Here, we report an analysis of variation in the prevalence and cytochrome b lineage distribution of avian malaria infection with respect to environmental and host factors, and their interactions, in a single blue tit (Cyanistes caeruleus) population. Of 11 Plasmodium and Haemoproteus cytochrome b lineages found in 997 breeding individuals, the three most numerous (pSGS1, pTURDUS1 and pBT7) were considered separately, in addition to analyses of all avian malaria lineages pooled. Our analyses revealed marked spatial differences in the prevalence and distribution of these lineages, with local prevalence of malaria within the population ranging from over 60% to less than 10%. In addition, we found several more complex patterns of prevalence with respect to local landscape features, host state, parasite genotype, and their interactions. We discuss the implications of such heterogeneity in parasite infection at a local scale for the study of the ecology and evolution of infectious diseases in natural populations. The increased resolution afforded by the combination of molecular genetic and geographical information systems (GIS) tools has the potential to provide many insights into the epidemiology, evolution and ecology of these parasites in the future.

Genetic characterization of avian malaria (Protozoa) in the endangered lesser kestrel, Falco naumanni

We genetically analyzed avian malaria (Protozoa) isolated from lesser kestrels (Falco naumanni) breeding in La Mancha, Central Spain. A total of 586 adult individuals were screened for blood parasites using a very efficient polymerase chain reaction approach that amplifies a partial segment (498 bp) of the cytochrome b gene of avian malaria of the genera Haemoproteus and Plasmodium. The prevalence of Plasmodium was 8.2%, and the prevalence of Haemoproteus was 4.1%. Sequence analyses revealed six unique lineages of avian malaria, three Plasmodium (LK5, LK6, RTSR1) and three Haemoproteus (LK2, LK3, LK4). According to sequence divergence, these lineages seem to correspond to at least three different species, although all recovered lineages could be independent evolutionary units. The third most common lineage (RTSR1) has been previously retrieved from two other avian host species, including a resident African bird species and a trans-Saharan migrant passerine, suggesting that lesser kestrels could acquire this Plasmodium lineage at their winter quarters in Africa.

The hitchhiker's guide to avian malaria

The ecological mechanisms underlying the dispersal of parasites are poorly understood, which is of particular concern in view of currently emerging infectious diseases. In a new study, Pérez-Tris and Bensch examined the distribution and prevalence of avian malaria in a migratory bird across Western Europe. They concluded that repeated independent evolution of year-round transmission has enabled some avian malaria lineages to become more widespread, and more prevalent, than lineages that are transmitted only during the summer. This study blurs the boundaries of evolutionary ecology, epidemiology and macroecology with great potential for cross-disciplinary research.

A phylogenetic comparison of gene trees constructed from plastid, mitochondrial and genomic DNA of Plasmodium species

Gene trees of Plasmodium species have been reported for the nuclear encoded genes (e.g. the Small Subunit rRNA) and a mitochondrial encoded gene, cytochrome b. Here, we have analyzed a plastid gene coding for caseinolytic protease ClpC, whose structure, function and evolutionary history have been studied in various organisms. This protein possesses a 220–250 amino acid long AAA domain (ATPases associated with a variety of cellular activities) that belongs to the Walker super family of ATPases and GTPases. We have sequenced the AAA motif of this gene, encoding the protein from nine different species of Plasmodium infecting rodents, birds, monkeys, and humans. The codon usage and GC content of each gene were nearly identical in contrast to the widely varying nucleotide composition of genomic DNAs. Phylogenetic trees derived from both DNA and inferred protein sequences have consistent topologies. We have used the ClpC sequence to analyze the phylogenetic relationship among Plasmodium species and compared it with those derived from mitochondrial and genomic sequences. The results corroborate well with the trees constructed using the mitochondrially encoded cytochrome b. However, an important element distinguishes the trees: the placement of Plasmodium elongatum near the base of the plastid tree, indicating an ancient lineage of parasites in birds that branches from the tree prior to other lineages of avian malaria and the human parasite, P. falciparum.

Human malaria parasites

Plasmodium relictum is a widespread invasive agent of avian malaria, responsible for acute, chronic and debilitating diseases in many species of birds. Recent PCR-based studies revealed astonishing genetic diversity of avian malaria parasites (genus Plasmodium), with numerous genetic lineages deposited in GenBank. Many studies addressed distribution and evolutionary relationships of avian Plasmodium lineages, but information about patterns of development of different lineages in mosquito vectors remains insufficient. Here we present data on sporogonic development of 2 widespread mitochondrial cytochrome b lineages (cyt b) of P. relictum (pSGS1 and pGRW11) in mosquito Culex pipiens pipiens. Genetic distance between these lineages is 0.2%; they fall in a well-supported clade in the phylogenetic tree. Three P. relictum strains were isolated from common crossbill (Loxia curvirostra, lineage pSGS1), domestic canary (Serinus canaria domestica, pSGS1) and house sparrow (Passer domesticus, pGRW11). These strains were multiplied in domestic canaries and used as donors of malarial gametocytes to infect C. p. pipiens. Mosquitoes were allowed to take blood meal on infected canaries and then dissected on intervals to study development of sporogonic stages. All 3 strains developed synchronously and completed sporogony in this vector, with infective sporozoites reported in the salivary glands on the day 14 after infection. Ookinetes, oocysts and sporozoites of all strains were indistinguishable morphologically. This study shows that patterns of sporogonic development of the closely related lineages pSGS1 and pGRW11 and different strains of the lineage pSGS1 of P. relictum are similar indicating that phylogenetic trees based on the cyt b gene likely can be used for predicting sporogonic development of genetically similar avian malaria lineages in mosquito vectors.