Differences in host species relationships and biogeographic influences produce contrasting patterns of prevalence, community composition and genetic structure in two genera of avian malaria parasites in southern Melanesia

1. Host-parasite interactions have the potential to influence broadscale ecological and evolutionary processes, levels of endemism, divergence patterns and distributions in host populations. Understanding the mechanisms involved requires identification of the factors that shape parasite distribution and prevalence. 2. A lack of comparative information on community-level host-parasite associations limits our understanding of the role of parasites in host population divergence processes. Avian malaria (haemosporidian) parasites in bird communities offer a tractable model system to examine the potential for pathogens to influence evolutionary processes in natural host populations. 3. Using cytochrome b variation, we characterized phylogenetic diversity and prevalence of two genera of avian haemosporidian parasites, Plasmodium and Haemoproteus, and analysed biogeographic patterns of lineages across islands and avian hosts, in southern Melanesian bird communities to identify factors that explain patterns of infection. 4. Plasmodium spp. displayed isolation-by-distance effects, a significant amount of genetic variation distributed among islands but insignificant amounts among host species and families, and strong local island effects with respect to prevalence. Haemoproteus spp. did not display isolation-by-distance patterns, showed marked structuring of genetic variation among avian host species and families, and significant host species prevalence patterns. 5. These differences suggest that Plasmodium spp. infection patterns were shaped by geography and the abiotic environment, whereas Haemoproteus spp. infection patterns were shaped predominantly by host associations. Heterogeneity in the complement and prevalence of parasite lineages infecting local bird communities likely exposes host species to a mosaic of spatially divergent disease selection pressures across their naturally fragmented distributions in southern Melanesia. Host associations for Haemoproteus spp. indicate a capacity for the formation of locally co-adapted host-parasite relationships, a feature that may limit intraspecific gene flow or range expansions of closely related host species.

Natural malaria infection reduces starvation resistance of nutritionally stressed mosquitoes.

In disease ecology, there is growing evidence that environmental quality interacts with parasite and host to determine host susceptibility to an infection. Most studies of malaria parasites have focused on the infection costs incurred by the hosts, and few have investigated the costs on mosquito vectors. The interplay between the environment, the vector and the parasite has therefore mostly been ignored and often relied on unnatural or allopatric Plasmodium/vector associations. Here, we investigated the effects of natural avian malaria infection on both fecundity and survival of field-caught female Culex pipiens mosquitoes, individually maintained in laboratory conditions. We manipulated environmental quality by providing mosquitoes with different concentrations of glucose-feeding solution prior to submitting them to a starvation challenge. We used molecular-based methods to assess mosquitoes' infection status. We found that mosquitoes infected with Plasmodium had lower starvation resistance than uninfected ones only under low nutritional conditions. The effect of nutritional stress varied with time, with the difference of starvation resistance between optimally and suboptimally fed mosquitoes increasing from spring to summer, as shown by a significant interaction between diet treatment and months of capture. Infected and uninfected mosquitoes had similar clutch size, indicating no effect of infection on fecundity. Overall, this study suggests that avian malaria vectors may suffer Plasmodium infection costs in their natural habitat, under certain environmental conditions. This may have major implications for disease transmission in the wild.

The chitinase PfCHT1 from the human malaria parasite Plasmodium falciparum lacks proenzyme and chitin-binding domains and displays unique substrate preferences

Within hours after the ingestion of a blood meal, the mosquito midgut epithelium synthesizes a chitinous sac, the peritrophic matrix. Plasmodium ookinetes traverse the peritrophic matrix while escaping the mosquito midgut. Chitinases (EC 3.2.1.14) are critical for parasite invasion of the midgut: the presence of the chitinase inhibitor, allosamidin, in an infectious blood meal prevents oocyst development. A chitinase gene, PgCHT1, recently has been identified in the avian malaria parasite P. gallinaceum. We used the sequence of PgCHT1 to identify a P. falciparum chitinase gene, PfCHT1, in the P. falciparum genome database. PfCHT1 differs from PgCHT1 in that the P. falciparum gene lacks proenzyme and chitin-binding domains. PfCHT1 was expressed as an active recombinant enzyme in Escherichia coli. PfCHT1 shares with PgCHT1 a substrate preference unique to Plasmodium chitinases: the enzymes cleave tri- and tetramers of GlcNAc from penta- and hexameric oligomers and are unable to cleave smaller native chitin oligosaccharides. The pH activity profile of PfCHT1 and its IC50 (40 nM) to allosamidin are distinct from endochitinase activities secreted by P. gallinaceum ookinetes. Homology modeling predicts that PgCHT1 has a novel pocket in the catalytic active site that PfCHT1 lacks, which may explain the differential sensitivity of PfCHT1 and PgCHT1 to allosamidin. PfCHT1 may be the ortholog of a second, as yet unidentified, chitinase gene of P. gallinaceum. These results may allow us to develop novel strategies of blocking human malaria transmission based on interfering with P. falciparum chitinase.

Bird malaria,

Plates V, VI and XI accompanied by guard sheet with descriptive letterpress.

Emerging Infectious Disease: Host and Parasite Perspectives

Avian malaria and related haematozoa are nearly ubiquitous parasites that can impose fitness costs of variable severity and may, in some cases, cause substantial mortality in their host populations. One example of the latter, the emergence of avian malaria in the endemic avifauna of Hawaii, has become a model for understanding the consequences of human-mediated disease introduction. The drastic declines of native Hawaiian birds due to avian malaria provided the impetus for examining more closely several aspects of host-parasite interactions in this system. Host-specificity is an important character determining the extent to which a parasite may emerge. Traditional parasite classification, however, has used host information as a character in taxonomical identification, potentially obscuring the true host range of many parasites. To improve upon previous methods, I first developed molecular tools to identify parasites infecting a particular host. I then used these molecular techniques to characterize host-specificity of parasites in the genera Plasmodium and Haemoproteus. I show that parasites in the genus Plasmodium exhibit low specificity and are therefore most likely to emerge in new hosts in the future. Subsequently, I characterized the global distribution of the single lineage of P. relictum that has emerged in Hawaii. I demonstrate that this parasite has a broad host distribution worldwide, that it is likely of Old World origin and that it has been introduced to numerous islands around the world, where it may have been overlooked as a cause of decline in native birds. I also demonstrate that morphological classification of P. relictum does not capture differences among groups of parasites that appear to be reproductively isolated based on molecular evidence. Finally, I examined whether reduced immunological capacity, which has been proposed to explain the susceptibility of Hawaiian endemics, is a general feature of an "island syndrome" in isolated avifauna of the remote Pacific. I show that, over multiple time scales, changes in immune response are not uniform and that observed changes probably reflect differences in genetic diversity, parasite exposure and life history that are unique to each species.

Alterações hematológicas, bioquímicas e histopatológicas no modelo de malária aviária Gallus gallus por Plasmodium gallinaceum: papel do óxido nítrico

Malária é uma das doenças infecciosas de maior causa de morte no mundo. Modelos experimentais são necessários para melhor compreensão de mecanismos envolvidos na patogênese de doenças e desenvolvimento de novos tratamentos. Galinhas infectadas com Plasmodium gallinaceum fornecem bom modelo de malária devido a proximidade filogenética com o Plasmodium de humano assim como aspectos clínicos comuns, como a malária cerebral. O presente estudo objetivou investigar a participação do óxido nítrico no desenvolvimento da malária aviária, através do tratamento ou não com aminoguanidina (AG - inibidor da enzima Óxido Nítrico Sintase) in vivo de galinhas infectadas experimentalmente com P. gallinaceum. Foi verificado sobrevida, hematologia clássica, bioquímica sérica e patologia nos animais no percurso da infecção. Observou-se maior sobrevida nos animais tratados com AG, apesar de parasitemias mais elevadas. Houve ainda diminuição nos parâmetros hematológicos e aumento no Volume Corpuscular Médio de hemácias, indicando resposta medular para anemia. Linfopenia e trombocitopenia foram detectadas em animais infectados, com menor proporção nos animais tratados. Monócitos, linfócitos e heterófilos apresentaram aumento de tamanho e alterações que indicam ativação. Trombócitos também aumentaram de tamanho durante a infecção e apresentaram morfologia atípica. Os animais tratados mostraram lesões mais brandas nas secções histopatológicas de cérebro, fígado e baço, além de produção diminuída de NO, mesmo em alta parasitemia, em relação aos animais não tratados. Esses resultados confirmam a participação do mediador químico óxido nítrico na patogênese da malária no modelo experimental aviário.

Papel do óxido nítrico na infecção malárica por P. gallinaceum

Malária é uma das mais incidentes doenças infecciosas do mundo. Na Amazônia existem muitos casos de malária causados principalmente por duas espécies de protozoários, o Plasmodium vivax e o Plasmodium falciparum, sendo este último responsável pela maioria dos casos de malária grave, que geralmente levam a morte devido ao acometimento de múltiplos órgãos, como o cérebro. Um dos mediadores químicos amplamente estudados nessa patogênese é o Óxido Nítrico (NO), o qual apresenta papel controverso. Atualmente duas hipóteses principais são apontadas como potencializadoras na patogênese. Uma, que a MC é causa da superprodução de NO, produzido pela Óxido Nítrico Sintase Neuronal (nNOS), após um quadro de hipóxia. Outra, diz que a MC é a causa da resposta exacerbada do sistema imunológico com produção de NO pela Óxido Nítrico Sintase Induzida (iNOS), presente nos macrófagos quando ativados pro determinantes antigênicos. Devido grande relevância da doença e dificuldade em enteder a patologia, modelos experimentais têm sido estabelecidos com a finalidade de esclarecer vias potenciais da evolução para MC, dentre eles o modelo de malária aviária causada pelo Plasmodium gallinaceum. Pouco se sabe sobre o seu papel do NO em modelos de malária aviária, principalmente devido inexistência de marcadores específicos para avaliar expressão das enzimas de síntese. Diante disso é importante estabelecer protocolos de purificação da iNOS de galinhas para a produção de um possível marcador. Para tanto se faz necessário investigar o papel do NO durante a malária aviária, em modelo experimental in vivo e in vitro, com linhagens de macrófagos de galinha HD11. Animais infectados com P. gallinaceum tratados com aminoguanidina (AG), um inibidor da produção de NO, tiveram maior sobrevida, além de menores níveis de nitrito no plasma e em macrófagos derivados de monócitos do sangue periférico, sugerindo a inibição da iNOS. Nos experimentos in vitro, células HD11 tratadas com LPS mostraram produção aumentada de NO, inferindo aumento na expressão e atividade da iNOS. Na separação proteica, observamos padrões diferentes que podem ser associados a uma elevada expressão da iNOS nos macrófagos ativados com LPS. Esse estudo proporcionará o melhor entendimento do modelo de malária aviária em galinhas, incluindo a cerebral, e envolvimento do sistema nitrérgico em galinhas infectadas com P. gallinaceum.

Prevalence of haemosporidians in a Neotropical endemic bird area

Haemosporidians are vector-transmitted intracellular parasites that occur in many bird species worldwide and may have important implications for wild bird populations. Surveys of haemosporidians have traditionally focused on Europe and North America, and only recently have they been carried out in the Neotropics, where the prevalence and impacts of the disease have been less studied and are not well understood. In this study we carried out a survey in the endemic bird area of the Sierra Nevada de Santa Marta (SNSM), an isolated coastal massif in northern Colombia that contains a large number of biomes and that is experiencing high rates of habitat loss. We sampled birds from 25 species at 2 different altitudes (1640 and 2100 m asl) and determined avian haemosporidian infection by polymerase chain reaction and sequencing a portion of the cytochrome b (cyt b) gene of the parasite. From the sampled birds, 32.1% were infected by at least 1 of 12 unique cyt b lineages of haemosporidian genera: Plasmodium, Leucocytozoon, Haemoproteus, and subgenus Parahaemoproteus. We found a higher prevalence of avian haemosporidians at low altitudes (1640 m asl). All endemic bird species we sampled had at least one individual infected with avian haemosporidians. We also found evidence of higher overall prevalence among endemic rather than nonendemic birds, suggesting higher susceptibility in endemic birds. Overall, our findings suggest a high haemosporidian species richness in the bird community of the SNSM. Considering the rate of habitat loss that this area is experiencing, it is important to understand how avian haemosporidians affect bird populations; furthermore, more exhaustive sampling is required to fully comprehend the extent of avian haemosporidian infection in the area.

Label-free enrichment of avian Leucocytozoon using flow cytometric sorting

The group of haemosporidian parasites is of general interest to basic and applied science, since several species infect mammals, leading to malaria and associated disease symptoms. Although the great majority of haemosporidian parasites appear in bird hosts, as in the case of Leucocytozoon buteonis, there is little genomic information about genetic aspects of their co-evolution with hosts. Consequently, there is a high need for parasite-enrichment strategies enabling further analyses of the genomes, namely without exposure to DNA-intercalating dyes. Here, we used flow cytometry without an additional labelling step to enrich L. buteonis from infected buzzard blood. A specific, defined area of two-dimensional scattergramms was sorted and the fraction was further analysed. The successful enrichment of L. buteonis in the sorted fraction was demonstrated by Giemsa-staining and qPCR revealing a clear increase of parasite-specific genes, while host-specific genes were significantly decreased. This is the first report describing a labelling-free enrichment approach of L. buteonis from infected buzzard blood. The enrichment of parasites presented here is free of nucleic acid-intercalating dyes which may interfere with fluorescence-based methods or subsequent sequencing approaches.

Comparison of circumsporozoite proteins from avian and mammalian malarias: biological and phylogenetic implications.

The circumsporozoite (CS) protein of malaria parasites (Plasmodium) covers the surface of sporozoites that invade hepatocytes in mammalian hosts and macrophages in avian hosts. CS genes have been characterized from many Plasmodium that infect mammals; two domains of the corresponding proteins, identified initially by their conservation (region I and region II), have been implicated in binding to hepatocytes. The CS gene from the avian parasite Plasmodium gallinaceum was characterized to compare these functional domains to those of mammalian Plasmodium and for the study of Plasmodium evolution. The P. gallinaceum protein has the characteristics of CS proteins, including a secretory signal sequence, central repeat region, regions of charged amino acids, and an anchor sequence. Comparison with CS signal sequences reveals four distinct groupings, with P. gallinaceum most closely related to the human malaria Plasmodium falciparum. The 5-amino acid sequence designated region I, which is identical in all mammalian CS and implicated in hepatocyte invasion, is different in the avian protein. The P. gallinaceum repeat region consists of 9-amino acid repeats with the consensus sequence QP(A/V)GGNGG(A/V). The conserved motif designated region II-plus, which is associated with targeting the invasion of liver cells, is also conserved in the avian protein. Phylogenetic analysis of the aligned Plasmodium CS sequences yields a tree with a topology similar to the one obtained using sequence data from the small subunit rRNA gene. The phylogeny using the CS gene supports the proposal that the human malaria P. falciparum is significantly more related to avian parasites than to other parasites infecting mammals, although the biology of sporozoite invasion is different between the avian and mammalian species.

Space-time variation of malaria incidence in Yunnan province, China

Abstract Background Understanding spatio-temporal variation in malaria incidence provides a basis for effective disease control planning and monitoring. Methods Monthly surveillance data between 1991 and 2006 for Plasmodium vivax and Plasmodium falciparum malaria across 128 counties were assembled for Yunnan, a province of China with one of the highest burdens of malaria. County-level Bayesian Poisson regression models of incidence were constructed, with effects for rainfall, maximum temperature and temporal trend. The model also allowed for spatial variation in county-level incidence and temporal trend, and dependence between incidence in June–September and the preceding January–February. Results Models revealed strong associations between malaria incidence and both rainfall and maximum temperature. There was a significant association between incidence in June–September and the preceding January–February. Raw standardised morbidity ratios showed a high incidence in some counties bordering Myanmar, Laos and Vietnam, and counties in the Red River valley. Clusters of counties in south-western and northern Yunnan were identified that had high incidence not explained by climate. The overall trend in incidence decreased, but there was significant variation between counties. Conclusion Dependence between incidence in summer and the preceding January–February suggests a role of intrinsic host-pathogen dynamics. Incidence during the summer peak might be predictable based on incidence in January–February, facilitating malaria control planning, scaled months in advance to the magnitude of the summer malaria burden. Heterogeneities in county-level temporal trends suggest that reductions in the burden of malaria have been unevenly distributed throughout the province.

An examination of the predictors of blood donors' intentions to donate during two phases of an avian influenza outbreak

BACKGROUND: Data from prior health scares suggest that an avian influenza outbreak will impact on people’s intention to donate blood; however research exploring this is scarce. Using an augmented theory of planned behavior (TPB), incorporating threat perceptions alongside the rational decision-making components of the TPB, the current study sought to identify predictors of blood donors’ intentions to donate during two phases of an avian influenza outbreak. STUDY DESIGN AND METHODS: Blood donors (N = 172) completed an on-line survey assessing the standard TPB predictors as well as measures of threat perceptions from the health belief model (HBM; i.e., perceived susceptibility and severity). Path analyses examined the utility of the augmented TPB to predict donors’ intentions to donate during a low- and high-risk phase of an avian influenza outbreak. RESULTS: In both phases, the model provided a good fit to the data explaining 69% (low risk) and 72% (high risk) of the variance in intentions. Attitude, subjective norm, and perceived susceptibility significantly predicted donor intentions in both phases. Within the low-risk phase, gender was an additional significant predictor of intention, while in the high-risk phase, perceived behavioral control was significantly related to intentions. CONCLUSION: An augmented TPB model can be used to predict donors’ intentions to donate blood in a low-risk and a high-risk phase of an outbreak of avian influenza. As such, the results provide important insights into donors’ decision-making that can be used by blood agencies to maintain the blood supply in the context of an avian influenza outbreak.

African Burkitt Lymphoma: Age-Specific Risk and Correlations with Malaria Biomarkers

African Burkitt lymphoma is an aggressive B-cell, non-Hodgkin lymphoma linked to Plasmodium falciparum malaria. Malaria biomarkers related to onset of African Burkitt lymphoma are unknown. We correlated age-specific patterns of 2,602 cases of African Burkitt lymphoma (60% male, mean ± SD age = 7.1 ± 2.9 years) from Uganda, Ghana, and Tanzania with malaria biomarkers published from these countries. Age-specific patterns of this disease and mean multiplicity of P. falciparum malaria parasites, defined as the average number of distinct genotypes per positive blood sample based on the merozoite surface protein-2 assessed by polymerase chain reaction, were correlated and both peaked between 5 and 9 years. This pattern, which was strong and consistent across regions, contrasted parasite prevalence, which peaked at 2 years and decreased slightly, and geometric mean parasite density, which peaked between 2 and 3 years and decreased sharply. Our findings suggest that concurrent infection with multiple malaria genotypes may be related to onset of African Burkitt lymphoma.