Genomic variation in Plasmodium relictum (lineage SGS1) and its implications for avian malaria infection outcomes: insights from experimental infections and genome-wide analysis.

BACKGROUND: The globally transmitted avian malaria parasite Plasmodium relictum (lineage SGS1) has been found to infect hundreds of different bird species with differences in infection outcomes ranging from more or less latent to potentially mortal. However, to date basic knowledge about the links between genetic differentiation and variation in infection outcome within this single malaria parasite species is lacking. METHODS: In this study, two different isolates of SGS1, obtained in the wild from two different host species, were used to investigate differences in their development in the blood and virulence in the experimentally infected canaries. Simultaneously, 258 kb of the parasite genome was screened for genetic differences using parasite mRNA and compared between experimental groups. RESULTS: The two isolates showed differences in development and caused mortality as well as effects on the blood parameters of their hosts. Although previous studies using single genes have shown very limited within lineage genetic diversity in the European population of SGS1, 226 SNPs were found across 322 genes, which separated the two experimental groups with a total of 23 SNPs that were fixed in either of the experimental groups. Moreover, genetic variation was found within each experimental group, hinting that each avian malaria infection harbours standing genetic variation that might be selected during each individual infection episode. CONCLUSION: These results highlight extensive genetic variation within the SGS1 population that is transferred into individual infections, thus adding to the complexity of the infection dynamics seen in these host-parasite interactions. Simultaneously, the results open up the possibility of understanding how genetic variation within the parasite populations is linked to the commonly observed differences in infection outcomes, both in experimental settings and in the wild.

RNAscope in situ hybridization reveals microvascular sequestration of Plasmodium relictum pSGS1 blood stages but absence of exo-erythrocytic dormant stages during latent infection of Serinus canaria.

BACKGROUND: Birds chronically infected with avian malaria parasites often show relapses of parasitaemia after latent stages marked by absence of parasites in the peripheral circulation. These relapses are assumed to result from the activation of dormant exo-erythrocytic stages produced during secondary (post-erythrocytic) merogony of avian Plasmodium spp. Yet, there is no morphological proof of persistent or dormant tissue stages in the avian host during latent infections. This study investigated persistence of Plasmodium relictum pSGS1 in birds with latent infections during winter, with the goal to detect presumed persisting tissue stages using a highly sensitive RNAscope® in situ hybridization technology. METHODS: Fourteen domestic canaries were infected with P. relictum pSGS1 by blood-inoculation in spring, and blood films examined during the first 4 months post infection, and during winter and spring of the following year. After parasitaemia was no longer detectable, half of the birds were dissected, and tissue samples investigated for persisting tissue stages using RNAscope ISH and histology. The remaining birds were blood-checked and dissected after re-appearance of parasitaemia, and their tissues equally examined. RESULTS: Systematic examination of tissues showed no exo-erythrocytic stages in birds exhibiting latent infections by blood-film microscopy, indicating absence of dormant tissue stages in P. relictum pSGS1-infected canaries. Instead, RNAscope ISH revealed rare P. relictum blood stages in capillaries of various tissues and organs, demonstrating persistence of the parasites in the microvasculature. Birds examined after re-appearance of parasitemia showed higher numbers of P. relictum blood stages in both capillaries and larger blood vessels, indicating replication during early spring and re-appearance in the peripheral circulation. CONCLUSIONS: The findings suggest that persistence of P. relictum pSGS1 during latent infection is mediated by continuous low-level erythrocytic merogony and possibly tissue sequestration of infected blood cells. Re-appearance of parasitaemia in spring seems to result from increased erythrocytic merogony, therefore representing recrudescence and not relapse in blood-inoculated canaries. Further, the study highlights strengths and limitations of the RNAscope ISH technology for the detection of rare parasite stages in tissues, providing directions for future research on persistence and tissue sequestration of avian malaria and related haemosporidian parasites.

Avian malaria in a feral-pet pigeon: a case report.

BACKGROUND: Avian malaria is caused by diverse parasite species of the genus Plasmodium, and it affects various bird species. The occurrence of this disease in some wild bird species is sparsely documented due to the scarce availability of samples. Hence the pathogenicity in some hosts is not completely known. In addition, feral birds may act as reservoirs bridging the transmission cycle from wild migratory birds to domestic and zoo-kept bird species. CASE PRESENTATION: An owner of pigeons adopted a feral pigeon (Columba livia forma domestica) and housed it together with his other pet-pigeons. The bird died unexpectedly a few weeks after a surgical procedure and necropsy revealed a severely anaemic carcass, with pale organs and hydropericardium. Histopathologic analysis revealed inflammatory infiltrates in the lung and liver, and monocytes and Kupffer cells contained haemozoin pigment indicative of phagocytosis of Plasmodium-infected erythrocytes. A high erythrocytic infection rate of 18% was evident in tissues and blood vessels in various organs. Furthermore, the thyroid had masses classified as thyroid carcinomas. Immunohistochemistry with anti- Plasmodium falciparum HSP70 antibody revealed positive signals in erythrocytes and intravascular leucocytes. Further microscopy analysis using a Hemacolor-stained impression smear revealed a high parasitaemia with an asynchronous infection showing all erythrocytic stages. Molecular diagnosis by PCR identified Plasmodium relictum, lineage GRW11 as the aetiological agent. The bird presented died most likely due to an acute infection as evidenced by the high blood parasitaemia, leading to major erythrocyte destruction. Further analyses of feral pigeons (n = 22) did not reveal any additional cases of Plasmodium infections. CONCLUSION: This study reports the first mortality associated with P. relictum lineage GRW11. The study supports previous studies, suggesting that Plasmodium infections are not frequent in pigeons. Host conditions like immunosuppression due to the tumour may have influenced the infection outcome in this fatal case. Use of anti-P. falciparum HSP70 antibody for detection of P. relictum antigens for immune assays in blood and tissue samples will be a useful tool for future studies.

Changes in hypothalamic-pituitary-adrenal axis function, immunity, and glucose during acute Plasmodium relictum infection in house sparrows (Passer domesticus).

Hosts of the same species vary in physiological responses to the same parasite, and some groups of individuals can disproportionately affect disease dynamics; however, the underlying pathophysiology of host-parasite interactions is poorly understood in wildlife. We tested the hypothesis that the hypothalamic-pituitary-adrenal (HPA) axis mediates host resistance and tolerance to avian malaria during the acute phase of infection by evaluating whether individual variation in circulating glucocorticoids predicted resistance to avian malaria in a songbird. We experimentally inoculated wild-caught house sparrows (Passer domesticus) with naturally sourced Plasmodium relictum and quantified baseline and restraint-induced circulating corticosterone, negative feedback ability, cellular and humoral immune function, and baseline and restraint-induced glycemia, prior to and during acute malaria infection. During peak parasitemia, we also evaluated the expression of several liver cytokines that are established pathological hallmarks of malaria in mammals: two pro-inflammatory (IFN-γ and TNF-α) and two anti-inflammatory (IL-10 and TGF-ß). Although most of the host metrics we evaluated were not correlated with host resistance or tolerance to avian malaria, this experiment revealed novel relationships between malarial parasites and the avian immune system that further our understanding of the pathology of malaria infection in birds. Specifically, we found that: (1) TNF-α liver expression was positively correlated with parasitemia; (2) sparrows exhibited an anti-inflammatory profile during malaria infection; and (3) IFN-γ and circulating glucose were associated with several immune parameters, but only in infected sparrows. We also found that, during the acute phase of infection, sparrows increased the strength of corticosterone negative feedback at the level of the pituitary. In the context of our results, we discuss future methodological considerations and aspects of host physiology that may confer resistance to avian malaria, which can help inform conservation and rehabilitation strategies for avifauna at risk.

Immune gene expression in the mosquito vector Culex quinquefasciatus during an avian malaria infection.

Plasmodium relictum is the most widespread avian malaria parasite in the world. It is listed as one of the 100 most dangerous invasive species, having been responsible for the extinction of several endemic bird species, and the near-demise of several others. Here we present the first transcriptomic study focused on the effect of P. relictum on the immune system of its vector (the mosquito Culex quinquefasciatus) at different times post-infection. We show that over 50% of immune genes identified as being part of the Toll pathway and 30%-40% of the immune genes identified within the Imd pathway are overexpressed during the critical period spanning the parasite's oocyst and sporozoite formation (8-12 days), revealing the crucial role played by both these pathways in this natural mosquito-Plasmodium combination. Comparison of infected mosquitoes with their uninfected counterparts also revealed some unexpected immune RNA expression patterns earlier and later in the infection: significant differences in expression of several immune effectors were observed as early as 30 min after ingestion of the infected blood meal. In addition, in the later stages of the infection (towards the end of the mosquito lifespan), we observed an unexpected increase in immune investment in uninfected, but not in infected, mosquitoes. In conclusion, our work extends the comparative transcriptomic analyses of malaria-infected mosquitoes beyond human and rodent parasites and provides insights into the degree of conservation of immune pathways and into the selective pressures exerted by Plasmodium parasites on their vectors.

Genomic advances in the study of the mosquito vector during avian malaria infection.

Invertebrate host­parasite associations are one of the keystones in order to understand vector-borne diseases. The study of these specific interactions provides information not only about how the vector is affected by the parasite at the gene-expression level, but might also reveal mosquito strategies for blocking the transmission of the parasites. A very well-known vector for human malaria is Anopheles gambiae. This mosquito species has been the main focus for genomics studies determining essential key genes and pathways over the course of a malaria infection. However, to-date there is an important knowledge gap concerning other non-mammophilic mosquito species, for example some species from the Culex genera which may transmit avian malaria but also zoonotic pathogens such as West Nile virus. From an evolutionary perspective, these 2 mosquito genera diverged 170 million years ago, hence allowing studies in both species determining evolutionary conserved genes essential during malaria infections, which in turn might help to find key genes for blocking malaria cycle inside the mosquito. Here, we extensively review the current knowledge on key genes and pathways expressed in Anopheles over the course of malaria infections and highlight the importance of conducting genomic investigations for detecting pathways in Culex mosquitoes linked to infection of avian malaria. By pooling this information, we underline the need to increase genomic studies in mosquito­parasite associations, such as the one in Culex­Plasmodium, that can provide a better understanding of the infection dynamics in wildlife and reduce the negative impact on ecosystems.

Special issue: avian malaria.

Avian malaria parasites or haemosporidia are found in bird species worldwide. This special issue focuses on 3 most commonly studied genera: Haemoproteus, Plasmodium and Leucocytozoon. Seven research articles and reviews are provided to illustrate the breadth of knowledge of the diversity of avian malaria parasites in different regional habitats and across bird species, and the use of avian haemosporidian systems to examine host­parasite eco-evolutionary questions.

Surveillance of avian malaria and related haemoparasites in common terns (Sterna hirundo) on the Atlantic coast of South America.

Haemosporidia (Apicomplexa, Haemosporida) are protozoa that infect vertebrate blood cells and are transmitted by vectors. Among vertebrates, birds possess the greatest diversity of haemosporidia, historically placed in 3 genera: Haemoproteus, Leucocytozoon and Plasmodium, the causative agent of avian malaria. In South America, existing data on haemosporidia are spatially and temporally dispersed, so increased surveillance is needed to improve the determination and diagnosis of these parasites. During the non-breeding season in 2020 and 2021, 60 common terns (Sterna hirundo) were captured and bled as part of ongoing research on the population health of migratory birds on the Argentinian Atlantic coast. Blood samples and blood smears were obtained. Fifty-eight samples were screened for Plasmodium, Haemoproteus and Leucocytozoon, as well as for Babesia parasites by nested polymerase chain reaction and by microscopic examination of smears. Two positive samples for Plasmodium were found. The cytochrome b lineages detected in the present study are found for the first time, and are close to Plasmodium lineages found in other bird orders. The low prevalence (3.6%) of haemoparasites found in this research was similar to those reported for previous studies on seabirds, including Charadriiformes. Our findings provide new information about the distribution and prevalence of haemosporidian parasites from charadriiforms in the southernmost part of South America, which remains understudied.

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.

Prevalence and genetic diversity of avian haemosporidian parasites in islands within a mega hydroelectric dam in the Brazilian Amazon.

The Brazilian Amazon supports an extremely diverse avifauna and serves as the diversification center for avian malaria parasites in South America. Construction of hydroelectric dams can drive biodiversity loss by creating islands incapable of sustaining the bird communities found in intact forest sites. Besides anthropogenic actions, the presence of parasites can also influence the dynamics and structure of bird communities. Avian malaria (Plasmodium) and related haemosporidian parasites (Haemoproteus and Leucocytozoon) are a globally distributed group of protozoan parasites recovered from all major bird groups. However, no study to date has analyzed the presence of avian haemosporidian parasites in fragmented areas such as land bridge islands formed during artificial flooding following the construction of hydroelectric dams. The aim of this study is to assess the prevalence and molecular diversity of haemosporidians in bird communities inhabiting artificial islands in the area of the Balbina Hydroelectric Dam. The reservoir area covers 443,700 ha with 3546 islands on the left bank of the Uatumã River known to contain more than 400 bird species. We surveyed haemosporidian infections in blood samples collected from 445 understory birds, belonging to 53 species, 24 families, and 8 orders. Passeriformes represented 95.5% of all analyzed samples. We found a low overall Plasmodium prevalence (2.9%), with 13 positive samples (two Plasmodium elongatum and 11 Plasmodium sp.) belonging to eight lineages. Six of these lineages were previously recorded in the Amazon, whereas two of them are new. Hypocnemis cantator, the Guianan Warbling Antbird, represented 38.5% of all infected individuals, even though it represents only 5.6% of the sampled individuals. Since comparison with Plasmodium prevalence data prior to construction of Balbina is not possible, other studies in artificially flooded areas are imperative to test if anthropogenic flooding may disrupt vector-parasite relationships leading to low Plasmodium prevalence.

First evidence for development of Plasmodium relictum (Grassi and Feletti, 1891) sporozoites in the salivary glands of Culex modestus Ficalbi, 1889.

The competence of insect vectors to transmit diseases plays a key role in host-parasite interactions and in the dynamics of avian malaria and other haemosporidian infections (Apicomplexa, Haemosporida). However, the presence of parasite DNA in the body of blood-sucking insects does not always constitute evidence for their competence as vectors. In this study, we investigate the susceptibility of wild-caught mosquitoes (Culex spp.) to complete sporogony of Plasmodium relictum (cyt b lineage SGS1) isolated from great tits (Parus major L., 1758). Adult female mosquitoes were collected with a CO2 bait trap overnight. A set of 50 mosquitoes was allowed to feed for 3 h at night on a single great tit infected with P. relictum. This trial was repeated on 6 different birds. The bloodfed mosquitoes that survived (n = 68) were dissected within 1-2 days (for ookinetes, n = 10) and 10-33 days post infection (for oocysts and sporozoites, n = 58) in order to confirm the respective parasite stages in their organs. The experiment confirmed the successful development of P. relictum (cyt b lineage SGS1) to the stage of sporozoites in Culex pipiens L., 1758 (n = 27) and in Culex modestus (n = 2). Our study provides the first evidence that C. modestus is a competent vector of P. relictum isolated from great tits, suggesting that this mosquito species could also play a role in the natural transmission of avian malaria.

Plasmodium relictum infection in Culex quinquefasciatus (Culicidae) decreases diel flight activity but increases peak dusk flight activity.

BACKGROUND: Parasites are recognized for their ability to modify host physiology and behaviours in ways that increase parasite fitness. Protozoan parasites of the genus Plasmodium are a group of widespread vector-borne parasites of vertebrates, causing disease to a wide range of hosts, but most notably to human and avian hosts. METHODS: The hypothesis that infection with the avian malaria, Plasmodium relictum (GRW4 lineage) impacts flight activity in one of their natural vectors, Culex quinquefasciatus, was tested using both parasites and mosquitoes colonized from local populations in East-Central Texas, USA. Groups of Cx. quinquefasciatus were allowed to feed directly on canaries with active P. relictum infections and control canaries with no P. relictum exposure history. Additionally, how P. relictum sporozoite invasion of mosquito salivary glands impacts mosquito flight activity behaviour was tested using a Locomotor Activity Monitor for both control and infected females. Generalized linear mixed models were used to evaluate the influence of infection status on the response variables of flight activity (continuous) and probability of flight occurring (binomial). RESULTS: Infection status was a significant predictor of flight activity and flight probability and interactions between infection status and experimental period of infection as well as infection status and dusk were statistically significant predictors of flight activity. Plasmodium relictum infected mosquitoes had a mean flight activity of 3.10 and control mosquitoes had an overall mean flight activity of 3.13. DISCUSSION: Based on these results, avian malaria parasites increase the flight activity of these mosquitoes at hours known for peak host-seeking behaviour but decrease overall diel activity. CONCLUSION: Although the ramifications of this behavioural change for P. relictum transmission are unclear, these results provide additional empirical evidence suggesting that avian malaria can influence mosquito behaviour and modulate transmission potential.

Transcriptional response of individual Hawaiian Culex quinquefasciatus mosquitoes to the avian malaria parasite Plasmodium relictum.

BACKGROUND: Plasmodium parasites that cause bird malaria occur in all continents except Antarctica and are primarily transmitted by mosquitoes in the genus Culex. Culex quinquefasciatus, the mosquito vector of avian malaria in Hawai'i, became established in the islands in the 1820s. While the deadly effects of malaria on endemic bird species have been documented for many decades, vector-parasite interactions in avian malaria systems are relatively understudied. METHODS: To evaluate the gene expression response of mosquitoes exposed to a Plasmodium infection intensity known to occur naturally in Hawai'i, offspring of wild-collected Hawaiian Cx. quinquefasciatus were fed on a domestic canary infected with a fresh isolate of Plasmodium relictum GRW4 from a wild-caught Hawaiian honeycreeper. Control mosquitoes were fed on an uninfected canary. Transcriptomes of five infected and three uninfected individual mosquitoes were sequenced at each of three stages of the parasite life cycle: 24 h post feeding (hpf) during ookinete invasion; 5 days post feeding (dpf) when oocysts are developing; 10 dpf when sporozoites are released and invade the salivary glands. RESULTS: Differential gene expression analyses showed that during ookinete invasion (24 hpf), genes related to oxidoreductase activity and galactose catabolism had lower expression levels in infected mosquitoes compared to controls. Oocyst development (5 dpf) was associated with reduced expression of a gene with a predicted innate immune function. At 10 dpf, infected mosquitoes had reduced expression levels of a serine protease inhibitor, and further studies should assess its role as a Plasmodium agonist in C. quinquefasciatus. Overall, the differential gene expression response of Hawaiian Culex exposed to a Plasmodium infection intensity known to occur naturally in Hawai'i was low, but more pronounced during ookinete invasion. CONCLUSIONS: This is the first analysis of the transcriptional responses of vectors to malaria parasites in non-mammalian systems. Interestingly, few similarities were found between the response of Culex infected with a bird Plasmodium and those reported in Anopheles infected with human Plasmodium. The relatively small transcriptional changes observed in mosquito genes related to immune response and nutrient metabolism support conclusions of low fitness costs often documented in experimental challenges of Culex with avian Plasmodium.

Exo-erythrocytic development of Plasmodium matutinum (lineage pLINN1) in a naturally infected roadkill fieldfare Turdus pilaris.

BACKGROUND: Species of Plasmodium (Haemosporida, Plasmodiidae) are remarkably diverse haemoparasites. Information on genetic diversity of avian malaria pathogens has been accumulating rapidly, however exo-erythrocytic development of these organisms remains insufficiently addressed. This is unfortunate because, contrary to Plasmodium species parasitizing mammals, the avian malaria parasites undergo several cycles of exo-erythrocytic development, often resulting in damage of various organs. Insufficient knowledge on the exo-erythrocytic development in most described Plasmodium species precludes the understanding of mechanisms of virulence during avian malaria. This study extends information on the exo-erythrocytic development of bird malaria parasites. METHODS: A roadkill fieldfare (Turdus pilaris) was sampled in Switzerland and examined using pathologic, cytologic, histologic, molecular and microbiologic methods. Avian malaria was diagnosed, and erythrocytic and exo-erythrocytic stages of the parasite were identified using morphologic characteristics and barcode DNA sequences of the cytochrome b gene. The species-specific characteristics were described, illustrated, and pathologic changes were reported. RESULTS: An infection with Plasmodium matutinum lineage pLINN1 was detected. Parasitaemia was relatively low (0.3%), with all erythrocytic stages (trophozoites, meronts and gametocytes) present in blood films. Most growing erythrocytic meronts were markedly vacuolated, which is a species-specific feature of this parasite's development. Phanerozoites at different stages of maturation were seen in leukocytes, macrophages, and capillary endothelial cells in most organs examined; they were particularly numerous in the brain. Like the erythrocytic meronts, growing phanerozoites were markedly vacuolated. Conspicuous exo-erythrocytic development and maturation in leucocytes suggests that this fieldfare was not adapted to the infection and the parasite was capable to escape from cellular immunity. CONCLUSIONS: This is the first report of exo-erythrocytic development of the malaria parasite lineage pLINN1 during single infection and the first report of this lineage in the fieldfare. The findings of multiple phanerozoites in brain, skeletal muscle, and eye tissue in combination with signs of vascular blockage and thrombus formation strongly suggest an impaired vision and neuromuscular responsiveness as cause of the unexpected collision with a slowly moving car. Further studies on exo-erythrocytic stages of haemosporidian parasites are pivotal to understand the true level of populational damage of avian malaria in wild birds.

Complete avian malaria parasite genomes reveal features associated with lineage-specific evolution in birds and mammals.

Avian malaria parasites are prevalent around the world and infect a wide diversity of bird species. Here, we report the sequencing and analysis of high-quality draft genome sequences for two avian malaria species, Plasmodium relictum and Plasmodium gallinaceum We identify 50 genes that are specific to avian malaria, located in an otherwise conserved core of the genome that shares gene synteny with all other sequenced malaria genomes. Phylogenetic analysis suggests that the avian malaria species form an outgroup to the mammalian Plasmodium species, and using amino acid divergence between species, we estimate the avian- and mammalian-infective lineages diverged in the order of 10 million years ago. Consistent with their phylogenetic position, we identify orthologs of genes that had previously appeared to be restricted to the clades of parasites containing Plasmodium falciparum and Plasmodium vivax, the species with the greatest impact on human health. From these orthologs, we explore differential diversifying selection across the genus and show that the avian lineage is remarkable in the extent to which invasion-related genes are evolving. The subtelomeres of the P. relictum and P. gallinaceum genomes contain several novel gene families, including an expanded surf multigene family. We also identify an expansion of reticulocyte binding protein homologs in P. relictum, and within these proteins, we detect distinct regions that are specific to nonhuman primate, humans, rodent, and avian hosts. For the first time in the Plasmodium lineage, we find evidence of transposable elements, including several hundred fragments of LTR-retrotransposons in both species and an apparently complete LTR-retrotransposon in the genome of P. gallinaceum.

Rediscovery of Plasmodium (Huffia) huffi (Apicomplexa, Haemosporida): a lost lineage from toucans.

Literature data on toucans haemosporidians are scarce and all reports come from investigations in Brazil. Muniz et al. (Rev Bras Malariol 3: 339-356, Muniz et al., Rev Bras Malariol 3:339-356, 1951) and Muniz and Soares (Rev Bras Malar 611-617, Muniz J, Soares R de RL (1954) Nota sôbre um parasita do gênero Plasmodium encontrado no Ramphastos toco Müller, 1776, "Tucano-Açu", e diferente do Plasmodium huffi: Plasmodium pinottii n. sp. Rev Bras Malar 611 - 617.) described two Plasmodium species, P. huffi and P. pinottii, in Ramphastos toco. Later, Manwell and Sessler (J Protozol 18: 570-574, Manwell and Sessler, Malaria Parasites of Toucans J Protozol 18:570-574, 1971) established a new subspecies, P. nucleophilum toucani. In the last review on avian haemosporidians, Valkiunas (Valkiunas, Avian malaria parasites and other haemosporidia, CRC Press, New York, 2005) highlighted that P. huffi was insufficiently characterized, considering it a lost lineage. Also, the original description of P. huffi was considered insufficiently clear, due to a possible co-infection of the toucan hosts with a Novyella-like species. Here, we redescribed the species Plasmodium (Huffia) huffi based on morphological and molecular data, which were found in two toucan species, Ramphastos toco and Pteroglossus aracari from Brazil. Morphological features of the specimens are markedly the same as the original description. In R. toco, we observe two individuals infected, one infected only with P. huffi and one co-infected with P. huffi and the Novyella-like species, as observed in the original description. Also, we observe one R. toco infected only with the Novyella-like species, identified by morphological and molecular data as Plasmodium (Novyella) nucleophilum nucleophilum. In this way, it was possible to redescribe Plasmodium huffi in detail, without the doubt characters observed in the original description. Moreover, by applying species delimitation algorithms to a large Plasmodium phylogeny, we were able to identify new possible hosts for P. huffi and extend its geographic distribution to include North America.

Treatment with chloroquine is retinotoxic in captive African penguins (Speniscus demersus). Attenuation and recovery of electroretinographic responses.

PURPOSE: To evaluate the effect of prophylactic anti-malarial chloroquine treatment, and its cessation, on electroretinographic (ERG) responses of captive African penguins. METHODS: A brief ERG protocol ("QuickRetCheck") was recorded under mesopic conditions with manual restraint and no sedation or pupil dilation. Birds were recorded on two separate occasions, first while being treated with a daily chloroquine dose of 10 mg/kg for 12 days (n = 15, treatment group) and second after 4 months without chloroquine treatment (n = 6, off-treatment group). Three birds were recorded on both occasions. Three other birds from the flock that died were studied histopathologically. RESULTS: Scotopic responses were unmeasurable in either recording and therefore were not analyzed. Mean a- and b-wave amplitudes of the mixed rod-cone responses to standard (3 cd·s/m2 ) and high (10 cd·s/m2 ) intensity flashes were higher in the off-treatment group. No difference in implicit times was observed. Sex, age, and number of previous chloroquine treatments did not affect ERG responses. Histopathology revealed Plasmodium spp.in the lungs, liver, and brain, but not in the eyes, of the necropsied birds, and there were no signs of retinitis or retinopathy. CONCLUSIONS: Daily chloroquine treatment was associated with attenuated ERG responses in penguins, which improve following cessation of treatment. Further work is warranted to establish a chloroquine dose that is efficacious yet has minimal adverse effects. Our results suggest that ERG responses of captive penguins undergoing ERG for any indication (such as prior to cataract surgery), must be evaluated in light of the birds' anti-malaria treatment status.

Host Transcriptional Responses to High- and Low-Virulent Avian Malaria Parasites.

The transcriptional response of hosts to genetically similar pathogens can vary substantially, with important implications for disease severity and host fitness. A low pathogen load can theoretically elicit both high and low host responses, as the outcome depends on both the effectiveness of the host at suppressing the pathogen and the ability of the pathogen to evade the immune system. Here, we investigate the transcriptional response of Eurasian siskins (Spinus spinus) to two closely related lineages of the malaria parasite Plasmodium relictum. Birds were infected with either the high-virulent lineage P. relictum SGS1, the low-virulent sister lineage P. relictum GRW4, or sham-injected (controls). Blood samples for RNA sequencing were collected at four time points during the course of infection, totaling 76 transcriptomes from 19 birds. Hosts infected with SGS1 experienced up to 87% parasitemia and major transcriptome shifts throughout the infection, and multiple genes showed strong correlation with parasitemia. In contrast, GRW4-infected hosts displayed low parasitemia (maximum 0.7%) with a minor transcriptional response. We furthermore demonstrate that the baseline gene expression levels of hosts prior to infection were irrelevant as immunocompetence markers, as they could not predict future pathogen load. This study shows that the magnitude of the host transcriptional response can differ markedly from related parasites with different virulence, and it enables a better understanding of the molecular interactions taking place between hosts and parasites.

Dynamics of avian haemosporidian assemblages through millennial time scales inferred from insular biotas of the West Indies.

Although introduced hemosporidian (malaria) parasites (Apicomplexa: Haemosporida) have hastened the extinction of endemic bird species in the Hawaiian Islands and perhaps elsewhere, little is known about the temporal dynamics of endemic malaria parasite populations. Haemosporidian parasites do not leave informative fossils, and records of population change are lacking beyond a few decades. Here, we take advantage of the isolation of West Indian land-bridge islands by rising postglacial sea levels to estimate rates of change in hemosporidian parasite assemblages over a millennial time frame. Several pairs of West Indian islands have been connected and separated by falling and rising sea levels associated with the advance and retreat of Pleistocene continental glaciers. We use island isolation following postglacial sea-level rise, ca. 2.5 ka, to characterize long-term change in insular assemblages of hemosporidian parasites. We find that assemblages on formerly connected islands are as differentiated as assemblages on islands that have never been connected, and both are more differentiated than local assemblages sampled up to two decades apart. Differentiation of parasite assemblages between formerly connected islands reflects variation in the prevalence of shared hemosporidian lineages, whereas differentiation between islands isolated by millions of years reflects replacement of hemosporidian lineages infecting similar assemblages of avian host species.

Presence and diversity of mixed avian Plasmodium spp. infections in introduced birds whose distribution overlapped with threatened New Zealand endemic birds.

Aims: To determine the presence of infection and co-infection of Plasmodium lineages in introduced birds at translocation sites for the North Island saddleback (Philesturnus rufusater), to investigate their role as Plasmodium spp. reservoirs.Methods: Blood samples were collected from introduced bird species, with a special focus on blackbirds (Turdus merula) and song thrushes (Turdus philomelos), at six locations in the North Island of New Zealand that were the origin, or translocation sites, for North Island saddleback. Where available, blood smears were examined, and blood samples were tested using nested PCR with subsequent sequence analysis, for the presence of Plasmodium spp.Results: Of the 55 samples tested using PCR analysis, 39 (71%) were positive for Plasmodium spp., and 28/40 (62%) blood smears were positive for Plasmodium spp. Overall, 31 blood samples were from blackbirds with 28/31 (90%) samples positive for Plasmodium spp. Six distinct avian Plasmodium lineages were identified, including three cosmopolitan lineages; Plasmodium vaughani SYAT05 was detected in 16 samples, Plasmodium matutinum Linn1 in 10 samples and Plasmodium elongatum GRW6 in eight samples. Mixed infections with more than one lineage were detected in 12 samples. Samples from two Australian magpies (Gymnorhina tibicen) were positive for Plasmodium. sp. lineage MYNA02, previously not identified in New Zealand.Conclusions and clinical relevance: This is the first report from New Zealand in which specific Plasmodium spp. mixed infections have been found in introduced birds. Co-infections with several cosmopolitan Plasmodium lineages were identified, as well as the first report in New Zealand of an exotic avian Plasmodium sp. lineage, in Australian magpies. Whilst the role of introduced birds in maintaining and spreading pathogenic avian malaria in New Zealand is unclear, there is a potential infection risk to native birds, especially where distributions overlap.