Gene expression reveals immune response strategies of naïve Hawaiian honeycreepers experimentally infected with introduced avian malaria.

The unprecedented rise in the number of new and emerging infectious diseases in the last quarter century poses direct threats to human and wildlife health. The introduction to the Hawaiian archipelago of Plasmodium relictum and the mosquito vector that transmits the parasite has led to dramatic losses in endemic Hawaiian forest bird species. Understanding how mechanisms of disease immunity to avian malaria may evolve is critical as climate change facilitates increased disease transmission to high elevation habitats where malaria transmission has historically been low and the majority of the remaining extant Hawaiian forest bird species now reside. Here, we compare the transcriptomic profiles of highly susceptible Hawai'i 'amakihi (Chlorodrepanis virens) experimentally infected with P. relictum to those of uninfected control birds from a naïve high elevation population. We examined changes in gene expression profiles at different stages of infection to provide an in-depth characterization of the molecular pathways contributing to survival or mortality in these birds. We show that the timing and magnitude of the innate and adaptive immune response differed substantially between individuals that survived and those that succumbed to infection, and likely contributed to the observed variation in survival. These results lay the foundation for developing gene-based conservation strategies for Hawaiian honeycreepers by identifying candidate genes and cellular pathways involved in the pathogen response that correlate with a bird's ability to recover from malaria infection.

Characterization of Plasmodium relictum, a cosmopolitan agent of avian malaria.

BACKGROUND: Microscopic research has shown that Plasmodium relictum is the most common agent of avian malaria. Recent molecular studies confirmed this conclusion and identified several mtDNA lineages, suggesting the existence of significant intra-species genetic variation or cryptic speciation. Most identified lineages have a broad range of hosts and geographical distribution. Here, a rare new lineage of P. relictum was reported and information about biological characters of different lineages of this pathogen was reviewed, suggesting issues for future research. METHODS: The new lineage pPHCOL01 was detected in Common chiffchaff Phylloscopus collybita, and the parasite was passaged in domestic canaries Serinus canaria. Organs of infected birds were examined using histology and chromogenic in situ hybridization methods. Culex quinquefasciatus mosquitoes, Zebra finch Taeniopygia guttata, Budgerigar Melopsittacus undulatus and European goldfinch Carduelis carduelis were exposed experimentally. Both Bayesian and Maximum Likelihood analyses identified the same phylogenetic relationships among different, closely-related lineages pSGS1, pGRW4, pGRW11, pLZFUS01, pPHCOL01 of P. relictum. Morphology of their blood stages was compared using fixed and stained blood smears, and biological properties of these parasites were reviewed. RESULTS: Common canary and European goldfinch were susceptible to the parasite pPHCOL01, and had markedly variable individual prepatent periods and light transient parasitaemia. Exo-erythrocytic and sporogonic stages were not seen. The Zebra finch and Budgerigar were resistant. Neither blood stages nor vector stages of all examined P. relictum lineages can be distinguished morphologically. CONCLUSION: Within the huge spectrum of vertebrate hosts, mosquito vectors, and ecological conditions, different lineages of P. relictum exhibit indistinguishable, markedly variable morphological forms. Parasites of same lineages often develop differently in different bird species. Even more, the variation of biological properties (parasitaemia dynamics, blood pathology, prepatent period) in different isolates of the same lineage might be greater than the variation in different lineages during development in the same species of birds, indicating negligible taxonomic value of such features. Available lineage information is excellent for parasite diagnostics, but is limited in predictions about relationships in certain host-parasite associations. A combination of experiments, field observations, microscopic and molecular diagnostics is essential for understanding the role of different P. relictum lineages in bird health.

Will a warmer and wetter future cause extinction of native Hawaiian forest birds?

Isolation of the Hawaiian archipelago produced a highly endemic and unique avifauna. Avian malaria (Plasmodium relictum), an introduced mosquito-borne pathogen, is a primary cause of extinctions and declines of these endemic honeycreepers. Our research assesses how global climate change will affect future malaria risk and native bird populations. We used an epidemiological model to evaluate future bird-mosquito-malaria dynamics in response to alternative climate projections from the Coupled Model Intercomparison Project. Climate changes during the second half of the century accelerate malaria transmission and cause a dramatic decline in bird abundance. Different temperature and precipitation patterns produce divergent trajectories where native birds persist with low malaria infection under a warmer and dryer projection (RCP4.5), but suffer high malaria infection and severe reductions under hot and dry (RCP8.5) or warm and wet (A1B) futures. We conclude that future global climate change will cause significant decreases in the abundance and diversity of remaining Hawaiian bird communities. Because these effects appear unlikely before mid-century, natural resource managers have time to implement conservation strategies to protect this unique avifauna from further decimation. Similar climatic drivers for avian and human malaria suggest that mitigation strategies for Hawai'i have broad application to human health.

Changing climate and the altitudinal range of avian malaria in the Hawaiian Islands - an ongoing conservation crisis on the island of Kaua'i.

Transmission of avian malaria in the Hawaiian Islands varies across altitudinal gradients and is greatest at elevations below 1500 m where both temperature and moisture are favorable for the sole mosquito vector, Culex quinquefasciatus, and extrinsic sporogonic development of the parasite, Plasmodium relictum. Potential consequences of global warming on this system have been recognized for over a decade with concerns that increases in mean temperatures could lead to expansion of malaria into habitats where cool temperatures currently limit transmission to highly susceptible endemic forest birds. Recent declines in two endangered species on the island of Kaua'i, the 'Akikiki (Oreomystis bairdi) and 'Akeke'e (Loxops caeruleirostris), and retreat of more common native honeycreepers to the last remaining high elevation habitat on the Alaka'i Plateau suggest that predicted changes in disease transmission may be occurring. We compared prevalence of malarial infections in forest birds that were sampled at three locations on the Plateau during 1994-1997 and again during 2007-2013, and also evaluated changes in the occurrence of mosquito larvae in available aquatic habitats during the same time periods. Prevalence of infection increased significantly at the lower (1100 m, 10.3% to 28.2%), middle (1250 m, 8.4% to 12.2%), and upper ends of the Plateau (1350 m, 2.0% to 19.3%). A concurrent increase in detections of Culex larvae in aquatic habitats associated with stream margins indicates that populations of the vector are also increasing. These increases are at least in part due to local transmission because overall prevalence in Kaua'i 'Elepaio (Chasiempis sclateri), a sedentary native species, has increased from 17.2% to 27.0%. Increasing mean air temperatures, declining precipitation, and changes in streamflow that have taken place over the past 20 years are creating environmental conditions throughout major portions of the Alaka'i Plateau that support increased transmission of avian malaria.

Environmental and geographical factors influence the occurrence and abundance of the southern house mosquito, Culex quinquefasciatus, in Hawai'i.

Hawaiian honeycreepers, a group of endemic Hawaiian forest birds, are being threatened by avian malaria, a non-native disease that is driving honeycreepers populations to extinction. Avian malaria is caused by the parasite Plasmodium relictum, which is transmitted by the invasive mosquito Culex quinquefasciatus. Environmental and geographical factors play an important role in shaping mosquito-borne disease transmission dynamics through their influence on the distribution and abundance of mosquitoes. We assessed the effects of environmental (temperature, precipitation), geographic (site, elevation, distance to anthropogenic features), and trap type (CDC light trap, CDC gravid trap) factors on mosquito occurrence and abundance. Occurrence was analyzed using classification and regression tree models (CART) and generalized linear models (GLM); abundance (count data) was analyzed using generalized linear mixed models (GLMMs). Models predicted highest mosquito occurrence at mid-elevation sites and between July and November. Occurrence increased with temperature and precipitation up to 580 mm. For abundance, the best model was a zero-inflated negative-binomial model that indicated higher abundance of mosquitoes at mid-elevation sites and peak abundance between August and October. Estimation of occurrence and abundance as well as understanding the factors that influence them are key for mosquito control, which may reduce the risk of forest bird extinction.

Linking avian malaria parasitemia estimates from quantitative PCR and microscopy reveals new infection patterns in Hawai'i.

Plasmodium parasites infect thousands of species and provide an exceptional system for studying host-pathogen dynamics, especially for multi-host pathogens. However, understanding these interactions requires an accurate assay of infection. Assessing Plasmodium infections using microscopy on blood smears often misses infections with low parasitemias (the fractions of cells infected), and biases in malaria prevalence estimates will differ among hosts that differ in mean parasitemias. We examined Plasmodium relictum infection and parasitemia using both microscopy of blood smears and quantitative polymerase chain reaction (qPCR) on 299 samples from multiple bird species in Hawai'i and fit models to predict parasitemias from qPCR cycle threshold (Ct) values. We used these models to quantify the extent to which microscopy underestimated infection prevalence and to more accurately estimate infection patterns for each species for a large historical study done by microscopy. We found that most qPCR-positive wild-caught birds in Hawaii had low parasitemias (Ct scores ≥35), which were rarely detected by microscopy. The fraction of infections missed by microscopy differed substantially among eight species due to differences in species' parasitemia levels. Infection prevalence was likely 4-5-fold higher than previous microscopy estimates for three introduced species, including Zosterops japonicus, Hawaii's most abundant forest bird, which had low average parasitemias. In contrast, prevalence was likely only 1.5-2.3-fold higher than previous estimates for Himatione sanguinea and Chlorodrepanis virens, two native species with high average parasitemias. Our results indicate that relative patterns of infection among species differ substantially from those observed in previous microscopy studies, and that differences depend on variation in parasitemias among species. Although microscopy of blood smears is useful for estimating the frequency of different Plasmodium stages and host attributes, more sensitive quantitative methods, including qPCR, are needed to accurately estimate and compare infection prevalence among host species.

Analysis of the trap gene provides evidence for the role of elevation and vector abundance in the genetic diversity of Plasmodium relictum in Hawaii.

BACKGROUND: The avian disease system in Hawaii offers an ideal opportunity to investigate host-pathogen interactions in a natural setting. Previous studies have recognized only a single mitochondrial lineage of avian malaria (Plasmodium relictum) in the Hawaiian Islands, but cloning and sequencing of nuclear genes suggest a higher degree of genetic diversity. METHODS: In order to evaluate genetic diversity of P. relictum at the population level and further understand host-parasite interactions, a modified single-base extension (SBE) method was used to explore spatial and temporal distribution patterns of single nucleotide polymorphisms (SNPs) in the thrombospondin-related anonymous protein (trap) gene of P. relictum infections from 121 hatch-year amakihi (Hemignathus virens) on the east side of Hawaii Island. RESULTS: Rare alleles and mixed infections were documented at three of eight SNP loci; this is the first documentation of genetically diverse infections of P. relictum at the population level in Hawaii. Logistic regression revealed that the likelihood of infection with a rare allele increased at low-elevation, but decreased as mosquito capture rates increased. The inverse relationship between vector capture rates and probability of infection with a rare allele is unexpected given current theories of epidemiology developed in human malarias. CONCLUSIONS: The results of this study suggest that pathogen diversity in Hawaii may be driven by a complex interaction of factors including transmission rates, host immune pressures, and parasite-parasite competition.

Reversion to virulence and efficacy of an attenuated Canarypox vaccine in Hawai'i 'Amakihi (Hemignathus virens).

Vaccines may be effective tools for protecting small populations of highly susceptible endangered, captive-reared, or translocated Hawaiian honeycreepers from introduced Avipoxvirus, but their efficacy has not been evaluated. An attenuated Canarypox vaccine that is genetically similar to one of two passerine Avipoxvirus isolates from Hawai'i and distinct from Fowlpox was tested to evaluate whether Hawai'i 'Amakihi (Hemignathus virens) can be protected from wild isolates of Avipoxvirus from the Hawaiian Islands. Thirty-one (31) Hawai'i 'Amakihi were collected from high-elevation habitats on Mauna Kea Volcano, where pox transmission is rare, and randomly divided into two groups. One group was vaccinated with Poximune C, whereas the other group received a sham vaccination with sterile water. Four of 15 (27%) vaccinated birds developed life-threatening disseminated lesions or lesions of unusually long duration, whereas one bird never developed a vaccine-associated lesion or "take." After vaccine lesions healed, vaccinated birds were randomly divided into three groups of five and challenged with either a wild isolate of Fowlpox (FP) from Hawai'i, a Hawai'i 'Amakihi isolate of a Canarypox-like virus (PV1), or a Hawai'i 'Amakihi isolate of a related, but distinct, passerine Avipoxvirus (PV2). Similarly, three random groups of five unvaccinated 'Amakihi were challenged with the same virus isolates. Vaccinated and unvaccinated 'Amakihi challenged with FP had transient infections with no clinical signs of infection. Mortality in vaccinated 'Amakihi challenged with PV1 and PV2 ranged from 0% (0/5) for PV1 to 60% (3/5) for PV2. Mortality in unvaccinated 'Amakihi ranged from 40% (2/5) for PV1 to 100% (5/5) for PV2. Although the vaccine provided some protection against PV1, both potential for vaccine reversion and low efficacy against PV2 preclude its use in captive or wild honeycreepers.

Transcriptome assembly and differential gene expression of the invasive avian malaria parasite Plasmodium relictum in Hawai'i.

The malaria parasite Plasmodium relictum (lineage GRW4) was introduced less than a century ago to the native avifauna of Hawai'i, where it has since caused major declines of endemic bird populations. One of the native bird species that is frequently infected with GRW4 is the Hawai'i 'amakihi (Chlorodrepanis virens). To achieve a better understanding of the transcriptional activities of this virulent parasite, we performed a controlled challenge experiment of 15 'amakihi that were infected with GRW4. Blood samples containing malaria parasites were collected at two time points (intermediate and peak infection stages) from host individuals that were either experimentally infected by mosquitoes or inoculated with infected blood. We then used RNA sequencing to assemble a high-quality blood transcriptome of P. relictum GRW4, allowing us to quantify parasite expression levels inside individual birds. We found few significant differences (one to two transcripts) in GRW4 expression levels between host infection stages and between inoculation methods. However, 36 transcripts showed differential expression levels among all host individuals, indicating a potential presence of host-specific gene regulation across hosts. To reduce the extinction risk of the remaining native bird species in Hawai'i, genetic resources of the local Plasmodium lineage are needed to enable further molecular characterization of this parasite. Our newly built Hawaiian GRW4 transcriptome assembly, together with analyses of the parasite's transcriptional activities inside the blood of Hawai'i 'amakihi, can provide us with important knowledge on how to combat this deadly avian disease in the future.

Experimental infection of Hawai'i 'Amakihi (hemignathus virens) with West Nile virus and competence of a co-occurring vector, culex quinquefasciatus: potential impacts on endemic Hawaiian avifauna.

Introduced mosquito-borne avian disease is a major limiting factor in the recovery and restoration of native Hawaiian forest birds. Annual epizootics of avian pox (Avipoxvirus) and avian malaria (Plasmodium relictum) likely led to the extinction of some species and continue to impact populations of susceptible Hawaiian honeycreepers (Drepanidinae). The introduction of a novel pathogen, such as West Nile virus (WNV), could result in further population declines and extinctions. During September and October 2004, we infected Hawai'i' Amakihi (Hemignathus virens) with a North American isolate of WNV by needle inoculation and mosquito bite to observe susceptibility, mortality, and illness in this endemic passerine, and to determine the vector competence of the co-occurring, introduced mosquito Culex quinquefasciatus. All experimentally infected Hawai'i ;Amakihi became viremic, with a mean titer >10(5) plaque-forming units (PFU)/ml, and they experienced clinical signs ranging from anorexia and lethargy to ataxia. The fatality rate among needle-inoculated Hawai'i' Amakihi (n=16) was 31.3%, but mortality in free-ranging birds is likely to increase due to predation, starvation, thermal stress, and concomitant infections of avian malaria and pox. Surviving Hawai'i' Amakihi seem to clear WNV from the peripheral blood by 7-10 days postinfection (DPI), and neutralizing antibodies were detected from 9 to 46 DPI. In transmission trials, Hawaiian Cx. quinquefasciatus proved to be a competent vector and Hawai'i Amakihi an adequate amplification host of WNV, suggesting that epizootic WNV could readily become an additional limiting factor of some native Hawaiian bird populations.

Population Differences in Susceptibility to Plasmodium relictum in Zebra Finches Taeniopygia guttata.

Domesticated Australian and Timor zebra finches (Taeniopygia guttata castanotis and Taeniopygia guttata guttata, respectively) were inoculated with canary (Serinus canaria) blood containing a Hawaiian isolate of Plasmodium relictum (lineage GRW04), a hemoparasite that causes avian malaria. In two experimental trials, TZFs but not AZFs developed parasitemia that was detected by microscopic examination of blood smears. In the second trial, in which molecular detection methods were used, a single AZF and five of six challenged TZFs were positive for the parasite. Additionally, P. relictum DNA was detected in multiple blood samples obtained from TZFs over the 28 days following challenge. TZFs may provide a useful, easily maintained, laboratory model for the study of Plasmodium interactions in passerines but are still inferior to canaries, the traditionally used model of avian malaria infection, in terms of supporting high-parasitemia infections.

Nota de investigación- Diferencias poblacionales en la susceptibilidad a Plasmodium relictum en diamantes cebra Taeniopygia guttata. Se inocularon diamantes cebra de Australia y de Timor (Taeniopygia guttata castanotis y Taeniopygia guttata guttata, respectivamente) con sangre de canario silvestre (Serinus canaria) que contenía un aislado hawaiano de Plasmodium relictum (linaje GRW04), que es un hemoparásito que causa la malaria aviar. En dos ensayos experimentales, los diamantes cebra de Timor desarrollaron una parasitemia detectada mediante un examen microscópico de frotis de sangre, pero los diamantes cebra australianos no desarrollaron dicha parasitemia. En el segundo ensayo, en el que se utilizaron métodos de detección molecular, un solo pinzón australiano y cinco de las seis aves de Timor desafiadas resultaron positivas para el parásito. Además, se detectó el ADN de P. relictum en múltiples muestras de sangre obtenidas de las aves de Timor durante 28 días posteriores al desafío. Los diamantes cebra de Timor pueden proporcionar un modelo de laboratorio útil y de fácil mantenimiento para el estudio de las interacciones de Plasmodium en passeriformes, pero áun son inferiores en comparación con los canarios, que son utilizados como modelo de infección por malaria aviar tradicionalmente usado en términos de apoyo a las infecciones con alta parasitemia.

Prevalence and distribution of pox-like lesions, avian malaria, and mosquito vectors in Kipahulu Valley, Haleakala National Park, Hawai'i, USA.

We determined prevalence and altitudinal distribution of introduced avian malarial infections (Plasmodium relictum) and pox-like lesions (Avipoxvirus) in forest birds from Kipahulu Valley, Haleakala National Park, on the island of Maui, and we identified primary larval habitat for the mosquito vector of this disease. This intensively managed wilderness area and scientific reserve is one of the most pristine areas of native forest remaining in the state of Hawai'i, and it will become increasingly important as a site for restoration and recovery of endangered forest birds. Overall prevalence of malarial infections in the valley was 8% (11/133) in native species and 4% (4/101) in nonnative passerines; prevalence was lower than reported for comparable elevations and habitats elsewhere in the state. Infections occurred primarily in 'Apapane (Himatione sanguinea) and Hawai'i 'Amakihi (Hemignathus virens) at elevations below 1,400 m. Pox-like lesions were detected in only two Hawai'i 'Amakihi (2%; 2/94) at elevations below 950 m. We did not detect malaria or pox in birds caught at 1,400 m in upper reaches of the valley. Adult mosquitoes (Culex quinquefasciatus) were captured at four sites at elevations of 640, 760, 915, and 975 m, respectively. Culex quinquefasciatus larvae were found only in rock holes along intermittent tributaries of the two largest streams in the valley, but not in standing surface water, pig wallows, ground pools, tree cavities, and tree fern cavities. Mosquito populations in the valley are low, and they are probably influenced by periods of high rainfall that flush stream systems.

Mitigating Future Avian Malaria Threats to Hawaiian Forest Birds from Climate Change.

Avian malaria, transmitted by Culex quinquefasciatus mosquitoes in the Hawaiian Islands, has been a primary contributor to population range limitations, declines, and extinctions for many endemic Hawaiian honeycreepers. Avian malaria is strongly influenced by climate; therefore, predicted future changes are expected to expand transmission into higher elevations and intensify and lengthen existing transmission periods at lower elevations, leading to further population declines and potential extinction of highly susceptible honeycreepers in mid- and high-elevation forests. Based on future climate changes and resulting malaria risk, we evaluated the viability of alternative conservation strategies to preserve endemic Hawaiian birds at mid and high elevations through the 21st century. We linked an epidemiological model with three alternative climatic projections from the Coupled Model Intercomparison Project to predict future malaria risk and bird population dynamics for the coming century. Based on climate change predictions, proposed strategies included mosquito population suppression using modified males, release of genetically modified refractory mosquitoes, competition from other introduced mosquitoes that are not competent vectors, evolved malaria-tolerance in native honeycreepers, feral pig control to reduce mosquito larval habitats, and predator control to improve bird demographics. Transmission rates of malaria are predicted to be higher than currently observed and are likely to have larger impacts in high-elevation forests where current low rates of transmission create a refuge for highly-susceptible birds. As a result, several current and proposed conservation strategies will be insufficient to maintain existing forest bird populations. We concluded that mitigating malaria transmission at high elevations should be a primary conservation goal. Conservation strategies that maintain highly susceptible species like Iiwi (Drepanis coccinea) will likely benefit other threatened and endangered Hawai'i species, especially in high-elevation forests. Our results showed that mosquito control strategies offer potential long-term benefits to high elevation Hawaiian honeycreepers. However, combined strategies will likely be needed to preserve endemic birds at mid elevations. Given the delay required to research, develop, evaluate, and improve several of these currently untested conservation strategies we suggest that planning should begin expeditiously.

Global phylogeographic limits of Hawaii's avian malaria.

The introduction of avian malaria (Plasmodium relictum) to Hawaii has provided a model system for studying the influence of exotic disease on naive host populations. Little is known, however, about the origin or the genetic variation of Hawaii's malaria and traditional classification methods have confounded attempts to place the parasite within a global ecological and evolutionary context. Using fragments of the parasite mitochondrial gene cytochrome b and the nuclear gene dihydrofolate reductase-thymidylate synthase obtained from a global survey of greater than 13000 avian samples, we show that Hawaii's avian malaria, which can cause high mortality and is a major limiting factor for many species of native passerines, represents just one of the numerous lineages composing the morphological parasite species. The single parasite lineage detected in Hawaii exhibits a broad host distribution worldwide and is dominant on several other remote oceanic islands, including Bermuda and Moorea, French Polynesia. The rarity of this lineage in the continental New World and the restriction of closely related lineages to the Old World suggest limitations to the transmission of reproductively isolated parasite groups within the morphological species.

Comparative susceptibility of introduced forest-dwelling mosquitoes in Hawai'i to avian malaria, Plasmodium relictum.

To identify potential vectors of avian malaria in Hawaiian native forests, the innate susceptibility of Aedes albopictus, Wyeomyia mitchellii, and Culex quinquefasciatus from 3 geographical sites along an altitudinal gradient was evaluated using local isolates of Plasmodium relictum. Mosquitoes were dissected 5-8 and 9-13 days postinfective blood meal and microscopically examined for oocysts and salivary-gland sporozoites. Sporogony was completed in all 3 species, but prevalence between species varied significantly. Oocysts were detected in 1-2% and sporozoites in 1-7% of Aedes albopictus that fed on infected ducklings. Wyeomyia mitchellii was slightly more susceptible, with 7-19% and 7% infected with oocysts and sporozoites, respectively. In both species, the median oocyst number was 5 or below. This is only the second Wyeomyia species reported to support development of a malarial parasite. Conversely, Culex quinquefasciatus from all 3 sites proved very susceptible. Prevalence of oocysts and sporozoites consistently exceeded 70%, regardless of gametocytemia or origin of the P. relictum isolate. In trials for which a maximum 200 oocysts were recorded, the median number of oocysts ranged from 144 to 200. It was concluded that Culex quinquefasciatus is the primary vector of avian malaria in Hawai'i.

A rapid method for counting nucleated erythrocytes on stained blood smears by digital image analysis.

Measures of parasitemia by intraerythrocytic hematozoan parasites are normally expressed as the number of infected erythrocytes per n erythrocytes and are notoriously tedious and time consuming to measure. We describe a protocol for generating rapid counts of nucleated erythrocytes from digital micrographs of thin blood smears that can be used to estimate intensity of hematozoan infections in nonmammalian vertebrate hosts. This method takes advantage of the bold contrast and relatively uniform size and morphology of erythrocyte nuclei on Giemsa-stained blood smears and uses ImageJ, a java-based image analysis program developed at the U.S. National Institutes of Health and available on the internet, to recognize and count these nuclei. This technique makes feasible rapid and accurate counts of total erythrocytes in large numbers of microscope fields, which can be used in the calculation of peripheral parasitemias in low-intensity infections.

PCR diagnostics underestimate the prevalence of avian malaria (Plasmodium relictum) in experimentally-infected passerines.

Several polymerase chain reaction (PCR)-based methods have recently been developed for diagnosing malarial infections in both birds and reptiles, but a critical evaluation of their sensitivity in experimentally-infected hosts has not been done. This study compares the sensitivity of several PCR-based methods for diagnosing avian malaria (Plasmodium relictum) in captive Hawaiian honeycreepers using microscopy and a recently developed immunoblotting technique. Sequential blood samples were collected over periods of up to 4.4 yr after experimental infection and rechallenge to determine both the duration and detectability of chronic infections. Two new nested PCR approaches for detecting circulating parasites based on P. relictum 18S rRNA genes and the thrombospondin-related anonymous protein (TRAP) gene are described. The blood smear and the PCR tests were less sensitive than serological methods for detecting chronic malarial infections. Individually, none of the diagnostic methods was 100% accurate in detecting subpatent infections, although serological methods were significantly more sensitive (97%) than either nested PCR (61-84%) or microscopy (27%). Circulating parasites in chronically infected birds either disappear completely from circulation or to drop to intensities below detectability by nested PCR. Thus, the use of PCR as a sole means of detection of circulating parasites may significantly underestimate true prevalence.

Experimental evidence for evolved tolerance to avian malaria in a wild population of low elevation Hawai'i 'Amakihi (Hemignathus virens).

Introduced vector-borne diseases, particularly avian malaria (Plasmodium relictum) and avian pox virus (Avipoxvirus spp.), continue to play significant roles in the decline and extinction of native forest birds in the Hawaiian Islands. Hawaiian honeycreepers are particularly susceptible to avian malaria and have survived into this century largely because of persistence of high elevation refugia on Kaua'i, Maui, and Hawai'i Islands, where transmission is limited by cool temperatures. The long term stability of these refugia is increasingly threatened by warming trends associated with global climate change. Since cost effective and practical methods of vector control in many of these remote, rugged areas are lacking, adaptation through processes of natural selection may be the best long-term hope for recovery of many of these species. We document emergence of tolerance rather than resistance to avian malaria in a recent, rapidly expanding low elevation population of Hawai'i 'Amakihi (Hemignathus virens) on the island of Hawai'i. Experimentally infected low elevation birds had lower mortality, lower reticulocyte counts during recovery from acute infection, lower weight loss, and no declines in food consumption relative to experimentally infected high elevation Hawai'i 'Amakihi in spite of similar intensities of infection. Emergence of this population provides an exceptional opportunity for determining physiological mechanisms and genetic markers associated with malaria tolerance that can be used to evaluate whether other, more threatened species have the capacity to adapt to this disease.