Classification for avian malaria parasite Plasmodium gallinaceum blood stages by using deep convolutional neural networks.

The infection of an avian malaria parasite (Plasmodium gallinaceum) in domestic chickens presents a major threat to the poultry industry because it causes economic loss in both the quality and quantity of meat and egg production. Computer-aided diagnosis has been developed to automatically identify avian malaria infections and classify the blood infection stage development. In this study, four types of deep convolutional neural networks, namely Darknet, Darknet19, Darknet19-448 and Densenet201 are used to classify P. gallinaceum blood stages. We randomly collected a dataset of 12,761 single-cell images consisting of three parasite stages from ten-infected blood films stained by Giemsa. All images were confirmed by three well-trained examiners. The study mainly compared several image classification models and used both qualitative and quantitative data for the evaluation of the proposed models. In the model-wise comparison, the four neural network models gave us high values with a mean average accuracy of at least 97%. The Darknet can reproduce a superior performance in the classification of the P. gallinaceum development stages across any other model architectures. Furthermore, the Darknet has the best performance in multiple class-wise classification, with average values of greater than 99% in accuracy, specificity, and sensitivity. It also has a low misclassification rate (< 1%) than the other three models. Therefore, the model is more suitable in the classification of P. gallinaceum blood stages. The findings could help us create a fast-screening method to help non-experts in field studies where there is a lack of specialized instruments for avian malaria diagnostics.

Experimental Malaria Infection Affects Songbirds' Nocturnal Migratory Activity.

Migratory animals encounter multiple parasite communities, raising concerns that migration may aid transport of infectious disease. How migration affects disease spread depends fundamentally on how disease affects migration, specifically whether infection alters individuals' migratory physiology and behavior. We inoculated white-throated sparrows (Zonotrichia albicollis) with avian malaria parasites (Plasmodium sp.), monitored parasite loads for 5 wk as the birds reached spring migratory condition, and compared nocturnal migratory restlessness (Zugunruhe), body composition (fat, lean, and whole-body mass), and hematocrit among experimentally infected birds, sham-inoculated birds, and birds that were exposed to parasites but resisted infection. Migratory restlessness increased over time in the study, but the rate of change varied between sham (control) birds, infected birds, and birds that resisted infection. We were unable to detect any effects of malaria exposure on body condition. Our findings suggest that encountering parasites affects migratory activity, regardless of whether infection occurs or is resisted.

Epidemiology, hematology, and unusual morphological characteristics of Plasmodium during an avian malaria outbreak in penguins in Brazil.

Avian malaria is a mosquito-borne disease caused by Plasmodium spp. protozoa, and penguins are considered particularly susceptible to this disease, developing rapid outbreaks with potentially high mortality. We report on an outbreak of avian malaria in Magellanic penguins (Spheniscus magellanicus) at a rehabilitation center in Espírito Santo, southeast Brazil. In August and September 2015, a total of 89 Magellanic penguins (87 juveniles and 2 adults) received care at Institute of Research and Rehabilitation of Marine Animals. Over a period of 2 weeks, Plasmodium infections were identified in eight individuals (9.0%), four of which died (mortality = 4.5%, lethality = 50%). Blood smears and sequencing of the mitochondrial cytochrome b gene revealed the presence of Plasmodium lutzi SPMAG06, Plasmodium elongatum GRW06, Plasmodium sp. PHPAT01, Plasmodium sp. SPMAG10, and Plasmodium cathemerium (sequencing not successful). Two unusual morphological features were observed in individuals infected with lineage SPMAG06: (a) lack of clumping of pigment granules and (b) presence of circulating exoerythrocytic meronts. Hematological results (packed cell volume, plasma total solids, complete blood cell counts) of positive individuals showed differences from those of negative individuals depending on the lineages, but there was no overarching pattern consistently observed for all Plasmodium spp. The epidemiology of the outbreak and the phylogeography of the parasite lineages detected in this study support the notion that malarial infections in penguins undergoing rehabilitation in Brazil are the result of the spillover inoculation by plasmodia that circulate in the local avifauna, especially Passeriformes.

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.

Lower Detection Probability of Avian Plasmodium in Blood Compared to Other Tissues.

We tested whether the probability of detecting avian haemosporidia (Plasmodium and Haemoproteus) using molecular techniques differs among blood, liver, heart, and pectoral muscle tissues. We used a paired design, sampling the 4 tissue types in 55 individuals of a wild South American suboscine antbird, the white-shouldered fire-eye (Pyriglena leucoptera). We also identified parasites to cytochrome b lineage. Detection probability was significantly lower in blood compared to the other 3 tissue types combined. Eight of 22 infections were not detected in blood samples; 4-7 infections were not detected in the other individual tissues. The same parasite lineage was recovered from different tissues.

Nest ecology of blood parasites in the European roller and its ectoparasitic carnid fly.

Haemosporidian parasites are considered the most important vector-borne parasites. However, vector identity and ecology is unknown for most such host-vector-parasite systems. In this study, we employ microscopic and molecular analyses to examine haemosporidian prevalence in a migratory, cavity-nesting bird, European roller Coracias garrulus, and its nidicolous blood-feeding ectoparasite Carnus hemapterus. This system is unique in that the ectoparasite is confined to a near-closed environment, in contrast to the free-wandering system of haematophagous dipterans such as mosquitoes. Blood film analysis confirms previous works in that Haemoproteus parasites are widely prevalent in adult rollers and belong to a single species, Haemoproteus coraciae. Leucocytozoon sp. and Trypanosoma sp. also are detected in adult rollers at low intensities with this technique. By means of molecular analysis, we report for the first time Plasmodium sp. presence in C. garrulus. Based on PCR results, Plasmodium parasites are relatively less prevalent than Haemoproteus parasites (20% vs. 31%) in rollers. In contrast, haemosporidian prevalences show the opposite trend for Carnus flies: Plasmodium sp. occurrence (62%) clearly predominates over that of Haemoproteus sp. (5%). A comparison between roller and Carnus samples reveals a significantly higher prevalence of Plasmodium sp. in Carnus samples. Insect survey and phylogenetic analysis suggest Culicoides flies as Haemoproteus sp. vectors, which appear to readily transmit the parasite in southern Spain. This study does not find support for Carnus flies to serve as biological or mechanical vectors of haemosporidians. In spite of this, nidicolous blood-feeding ectoparasites, such as carnid flies, appear as a suitable model for studies on the occurrence and temporal dynamics of avian haemosporidians such as Plasmodium sp. present at low intensities.

Avian malaria in a boreal resident species: long-term temporal variability, and increased prevalence in birds with avian keratin disorder.

The prevalence of vector-borne parasitic diseases is widely influenced by biological and ecological factors. Environmental conditions such as temperature and precipitation can have a marked effect on haemosporidian parasites (Plasmodium spp.) that cause malaria and those that cause other malaria-like diseases in birds. However, there have been few long-term studies monitoring haemosporidian infections in birds in northern latitudes, where weather conditions can be highly variable and the effects of climate change are becoming more pronounced. We used molecular methods to screen more than 2,000 blood samples collected from black-capped chickadees (Poecile atricapillus), a resident passerine bird. Samples were collected over a 10 year period, mostly during the non-breeding season, at seven sites in Alaska, USA. We tested for associations between Plasmodium prevalence and local environmental conditions including temperature, precipitation, site, year and season. We also evaluated the relationship between parasite prevalence and individual host factors of age, sex and presence or absence of avian keratin disorder. This disease, which causes accelerated keratin growth in the beak, provided a natural study system in which to test the interaction between disease state and malaria prevalence. Prevalence of Plasmodium infection varied by year, site, age and individual disease status but there was no support for an effect of sex or seasonal period. Significantly, birds with avian keratin disorder were 2.6 times more likely to be infected by Plasmodium than birds without the disorder. Interannual variation in the prevalence of Plasmodium infection at different sites was positively correlated with summer temperatures at the local but not statewide scale. Sequence analysis of the parasite cytochrome b gene revealed a single Plasmodium spp. lineage, P43. Our results demonstrate associations between prevalence of avian malaria and a variety of biological and ecological factors. These results also provide important baseline data that will be informative for predicting future changes in Plasmodium prevalence in the subarctic.

The avian transcriptome response to malaria infection.

Malaria parasites are highly virulent pathogens which infect a wide range of vertebrates. Despite their importance, the way different hosts control and suppress malaria infections remains poorly understood. With recent developments in next-generation sequencing techniques, however, it is now possible to quantify the response of the entire transcriptome to infections. We experimentally infected Eurasian siskins (Carduelis spinus) with avian malaria parasites (Plasmodium ashfordi), and used high-throughput RNA-sequencing to measure the avian transcriptome in blood collected before infection (day 0), during peak parasitemia (day 21 postinfection), and when parasitemia was decreasing (day 31). We found considerable differences in the transcriptomes of infected and uninfected individuals, with a large number of genes differentially expressed during both peak and decreasing parasitemia stages. These genes were overrepresented among functions involved in the immune system, stress response, cell death regulation, metabolism, and telomerase activity. Comparative analyses of the differentially expressed genes in our study to those found in other hosts of malaria (human and mouse) revealed a set of genes that are potentially involved in highly conserved evolutionary responses to malaria infection. By using RNA-sequencing we gained a more complete view of the host response, and were able to pinpoint not only well-documented host genes but also unannotated genes with clear significance during infection, such as microRNAs. This study shows how the avian blood transcriptome shifts in response to malaria infection, and we believe that it will facilitate further research into the diversity of molecular mechanisms that hosts utilize to fight malaria infections.

Testing for associations between hematozoa infection and mercury in wading bird nestlings.

Several wading bird species in the southeastern US have a history of infection by hematozoa/avian malaria as well as mercury accumulation through their diet, and thus may be exposed to two, generally sublethal, yet chronic, stressors. We analyzed nestling wading birds (n = 171) of varying size and trophic position from the southeastern US, and a smaller sample (n = 23) of older, free-ranging birds, to look for potential interrelationships between infection by hematozoa and mercury (Hg) uptake. Only one nestling was PCR positive for hematozoa (Plasmodium/Haemoproteus) whereas nine (39%) of the older wading birds were positive. Sequencing indicated that both nestling and adult positives were infected with Plasmodium. Given the low infection rate of the nestlings, there was no association between Hg and malaria. The older birds exhibited a possible malaria/Hg association, but it may be confounded by their greater potential exposure period and large-scale movements.

An inverse association between West Nile virus serostatus and avian malaria infection status.

BACKGROUND: Various ecological and physiological mechanisms might influence the probability that two or more pathogens may simultaneously or sequentially infect a host individual. Concurrent infections can have important consequences for host condition and fitness, including elevated mortality risks. In addition, interactions between coinfecting pathogens may have important implications for transmission dynamics. METHODS: Here, we explore patterns of association between two common avian pathogens (West Nile virus and avian malaria parasites) among a suburban bird community in Chicago, IL, USA that share mosquito vectors. We surveyed 1714 individual birds across 13 species for both pathogens through established molecular protocols. RESULTS: Field investigations of haemosporidian and West Nile virus (WNV) infections among sampled birds yielded an inverse association between WNV serostatus and Plasmodium infection status. This relationship occurred in adult birds but not in juveniles. There was no evidence for a relationship between Haemoproteus infection and WNV serostatus. We detected similar prevalence of Plasmodium among birds captured with active WNV infections and spatiotemporally paired WNV-naïve individuals of the same species, demonstrating that the two pathogens can co-infect hosts. CONCLUSIONS: Mechanisms explaining the negative association between WNV serostatus and Plasmodium infection status remain unclear and must be resolved through experimental infection procedures. However, our results highlight potential interactions between two common avian pathogens that may influence their transmission among hosts. This is especially relevant considering that West Nile virus is a common zoonotic pathogen with public health implications. Moreover, both pathogens are instructive models in infectious disease ecology, and infection with either has fitness consequences for their avian hosts.

Disease screening of three breeding populations of adult exhibition budgerigars (Melopsittacus undulatus) in New Zealand reveals a high prevalence of a novel polyomavirus and avian malaria infection.

Disease surveillance is vital to the management of New Zealand's endemic and threatened avian species. Three infectious agents that are potential threats to New Zealand's endemic birds include avian polyomavirus (APV), beak and feather disease virus (BFDV), and avian malaria. All three agents have been reported in New Zealand; however, possible reservoir populations have not been identified. In this communication, we report the first study of APV, BFDV, and avian malaria in introduced adult exhibition budgerigars (Melopsittacus undulatus) in New Zealand. Blood samples were collected from 90 living adult budgerigars from three breeding locations in the North Island of New Zealand. An overall APV prevalence of 22% was determined using a broad-spectrum nested PCR that amplified the major capsid protein VP1 gene of polyomavirus. Phylogenetic analysis of the VP1 gene revealed a unique isolate of APV, which had a sequence divergence of 32% to previously reported budgerigar fledgling disease strains and 33% to the recently reported New Zealand finch isolate. All of the budgerigars sampled were found to be PCR negative for BFDV, and an overall prevalence of 30% was detected by PCR for avian malaria. Sequencing revealed the presence of ubiquitous malarial strains and also the potentially destructive Plasmodium relictum strain. The results of this study suggest that both APV and avian malaria are present in New Zealand adult budgerigars, and our study highlights the need for further studies to determine whether these pathogens in captive bird populations may be a threat or spill over into New Zealand's endemic and threatened avifauna and whether prevention and control methods need to be implemented.

A transmission model for the ecology of an avian blood parasite in a temperate ecosystem.

Most of our knowledge about avian haemosporidian parasites comes from the Hawaiian archipelago, where recently introduced Plasmodiumrelictum has contributed to the extinction of many endemic avian species. While the ecology of invasive malaria is reasonably understood, the ecology of endemic haemosporidian infection in mainland systems is poorly understood, even though it is the rule rather than the exception. We develop a mathematical model to explore and identify the ecological factors that most influence transmission of the common avian parasite, Leucocytozoonfringillinarum (Apicomplexa). The model was parameterized from White-crowned Sparrow (Zonotrichialeucophrys) and S. silvestre / craigi black fly populations breeding in an alpine ecosystem. We identify and examine the importance of altricial nestlings, the seasonal relapse of infected birds for parasite persistence across breeding seasons, and potential impacts of seasonal changes in black fly emergence on parasite prevalence in a high elevation temperate system. We also use the model to identify and estimate the parameters most influencing transmission dynamics. Our analysis found that relapse of adult birds and young of the year birds were crucial for parasite persistence across multiple seasons. However, distinguishing between nude nestlings and feathered young of the year was unnecessary. Finally, due to model sensitivity to many black fly parameters, parasite prevalence and sparrow recruitment may be most affected by seasonal changes in environmental temperature driving shifts in black fly emergence and gonotrophic cycles.

The seroprevalence of avipoxvirus and its association with avian malaria (Plasmodium spp.) infection in introduced passerine birds in the southern regions of the North Island of New Zealand.

Blood samples were collected from 65 free-ranging birds from six species in the southern North Island of New Zealand. Sera from the birds were tested for the presence of avipoxvirus (APV) antibodies by enzyme-linked immunosorbent assay (ELISA), and blood cells from 55 birds were also tested for Plasmodium spp. by PCR. Forty-five birds (69.2%) tested seropositive to APV. Song thrushes (Turdus philomelos) presented the highest seroprevalence at 100% (4/4), followed by Eurasian blackbirds (Turdus merula) (96.86%, 31/32), chaffinches (Fringilla coelebs) (54.55%, 6/11), starlings (Sturnus vulgaris) (25%, 3/12), greenfinches (Carduelis chloris) (25%, 1/4), and European goldfinches (Carduelis carduelis) (0%, 0/2). Plasmodium spp. DNA was detected in 15/55 birds (27.3%), including 11 Eurasian blackbirds, one song thrush, and three starlings. Eight Eurasian blackbird isolates (73%) grouped within the subgenus Novyella. Two Eurasian blackbird isolates and the song thrush isolate clustered within a different group with previously reported lineages LINN1 and AFTRU5. In addition, all three starling isolates clustered within the well-characterized lineage Plasmodium (Huffia) elongatum GRW06. All Plasmodium-positive Eurasian blackbirds and the song thrush were seropositive to APV, whereas only 67% of Plasmodium-positive starlings showed evidence of previous exposure to APV. A significant relationship between birds seropositive to APV and birds infected by Plasmodium spp. was observed (chi2 = 5.69, df = 1, P = 0.0086). To the authors' knowledge this is the first report describing the seroprevalence of APV and its association with Plasmodium spp. infection in introduced bird species in New Zealand.

Parasitological and new molecular-phylogenetic characterization of the malaria parasite Plasmodium tejerai in South American penguins.

This study is the first report on mortality of Spheniscus magellanicus, penguin of South America, caused by Plasmodium tejerai, which was identified using morphological and molecular analyses. Blood stages (trophozoites, meronts and gametocytes) were reported and illustrated. The necropsy revealed marked splenomegaly and pulmonary edema, as well as moderate hepatomegaly and hydropericardium. The histopathology revealed the presence of tissue meronts in the macrophages and endothelial cells of multiple organs. The molecular analyses showed 5.6% of genetic divergence in cytochrome b gene between P. tejerai and Plasmodium relictum. Morphology of blood and tissue stages of P. tejerai is similar to P. relictum; the distinction between these two species requires experience in the identification of avian Plasmodium species. Molecular studies associated with reliably identified morphological species are useful for barcoding and comparisons with previous studies of wildlife malaria infections as well as for posterior phylogenetic and phylogeographic studies. S. magellanicus is a new host record of P. tejerai, which is the virulent parasite and worth more attention in avian conservation and veterinary medicine projects in South America.

Blood parasites in nestlings of wood stork populations from three regions of the American continent.

This study documents the prevalences and lineages of hemoparasites in wood stork nestlings from 3 regions of the American continent: southeastern United States (n = 90), northern Brazil (n = 74), and central-western Brazil (n = 125). Identification was based on PCR amplification of a mitochondrial small subunit ribosomal RNA gene. A fragment of the hemoparasite cytochrome B gene in infected individuals was utilized for Bayesian phylogenetic analysis. Four wood stork nestlings were infected by Haemoproteus, 1 from northern Brazil and 3 from the United States, and all shared the same haplotype. Morphological analysis confirmed the infection of the U.S. birds by Haemoproteus. Infection by Plasmodium was found in wood stork nestlings from northern (6) and central-western Brazil (14). Five Plasmodium lineages (MYCAMP1-2, and MYCAMP4-6) were found in the Brazilian central-western region and 3 Plasmodium lineages (MYCAMP2-3, and MYCAMP7) were found in the northern region. The most prevalent haplotype (MYCAMP2) differs from the others by 1 mutation, and the less prevalent haplotypes are derived from MYCAMP2. We did not find Plasmodium or Haemoproteus in nestlings younger than 15 and 30 days old, respectively. This is the first documentation of Plasmodium and Haemoproteus infection in wood storks in Brazilian breeding populations. Potential connectivity among wood stork populations was indirectly supported by the presence of identical Haemoproteus lineages in U.S. and northern Brazilian populations, and by the presence of identical Plasmodium haplotypes in the northern and central-western Brazilian populations.

Experimental inhibition of nitric oxide increases Plasmodium relictum (lineage SGS1) parasitaemia.

Malaria is a widespread vector-borne disease infecting a wide range of terrestrial vertebrates including reptiles, birds and mammals. In addition to being one of the most deadly infectious diseases for humans, malaria is a threat to wildlife. The host immune system represents the main defence against malaria parasites. Identifying the immune effectors involved in malaria resistance has therefore become a major focus of research. However, this has mostly involved humans and animal models (rodents) and how the immune system regulates malaria progression in non-model organisms has been largely ignored. The aim of the present study was to investigate the role of nitric oxide (NO) as an immune effector contributing to the control of the acute phase of infection with the avian malaria agent Plasmodium relictum. We used experimental infections of domestic canaries in conjunction with the inhibition of the enzyme inducible nitric oxide synthase (iNOS) to assess the protective function of NO during the infection, and the physiological costs paid by the host in the absence of an effective NO response. Our results show that birds treated with the iNOS inhibitor suffered from a higher parasitaemia, but did not pay a higher cost of infection (anaemia). While these findings confirm that NO contributes to the resistance to avian malaria during the acute phase of the infection, they also suggest that parasitaemia and costs of infection can be decoupled.

Plasmodium circumflexum in a Shikra (Accipiter badius): phylogeny and ultra-structure of the haematozoa.

A wild-caught, juvenile Shikra (Accipiter badius) was evaluated for rehabilitation at the Kasetsart University Raptor Rehabilitation Unit (KURRU) with a history of weakness. Plasmodium sp. was observed by both light and electron microscopy in blood obtained on day 1 of evaluation. Based on the appearance of erythrocytic meronts and gametocytes, the parasites were defined as Plasmodium (Giovannolaia) circumflexum. The sequence analysis of the mitochondrial cytochrome b gene from the plasmodia was closely related to parasites found in the Grey-headed woodpecker from Myanmar and the Brown hawk-owl from Singapore. Transmission electron microscopic examination revealed organelles in the haematozoa and heterophils that ingested the plasmodia. This is the first recorded case of Plasmodium circumflexum in a wild Shikra. This note emphasises the molecular characterisation and ultra-structure of the haematozoa.

A new morphologically distinct avian malaria parasite that fails detection by established polymerase chain reaction-based protocols for amplification of the cytochrome B gene.

Plasmodium polymorphum n. sp. (Haemosporida, Plasmodiidae) was found in the skylark, Alauda arvensis (Passeriformes: Alaudidae), during autumnal migration in southern Italy. This organism is illustrated and described based on the morphology of its blood stages. The most distinctive feature of this malaria parasite is the clear preference of its blood stages (trophozoites, meronts, and gametocytes) for immature red blood cells, including erythroblasts. Based on preference of erythrocytic meronts for immature red blood cells, P. polymorphum is most similar to species of the subgenus Huffia . This parasite can be readily distinguished from all other bird malaria parasites, including Plasmodium ( Huffia ) spp., due to preferential development and maturation of its gametocytes in immature red blood cells, a unique character for avian Plasmodium spp. In addition, the margins of nuclei in blood stages of P. polymorphum are markedly smooth and distinct; this is also a distinct diagnostic feature of this parasite. Plasmodium polymorphum has been recorded only in the skylark; it is probably a rare parasite, whose host range and geographical distribution remain unclear. Microscopic examination detected a light infection of Plasmodium relictum (lineage GRW11, parasitemia of <0.01%) in the same sample with P. polymorphum ; the latter parasite clearly predominated (3.5% parasitemia). However, experienced researchers were unable to detect sequences of mitochondrial cytochrome b gene (cyt b ) of P. polymorphum from the microscopically positive sample by using published and newly designed primers for DNA amplification of avian Plasmodium spp. The light parasitemia of P. relictum was easily detectable using several polymerase chain reaction (PCR)-based assays, but P. polymorphum was undetectable in all applied assays. Quantitative PCR also showed the presence of light parasitemia (0.06%) of the lineage GRW11 in this sample. This supports the conclusion that the morphologically distinct parasite observed along with P. relictum and predominant in the sample is genetically dissimilar from the lineage GRW11 based on cyt b sequence. In samples with co-infections, general PCR protocols tend to favor the amplification of the parasite with the higher parasitemia or the amplification with the best matching sequence to the primers. Because the parasitemia of P. polymorphum was >50-fold higher than that of P. relictum and several different primers were tested, we suggest that the failure to amplify P. polymorphum is a more complex problem than why co-infections are commonly overlooked in PCR-based studies. We suggest possible explanations of these results and call for additional research on evolution of mitochondrial genome of hemosporidian parasites.

A microfluidic platform to isolate avian erythrocytes infected with Plasmodium gallinaceum malaria parasites based on surface morphological changes.

This paper reports on a microfluidic platform to isolate and study avian red blood cells (RBCs) infected to various degrees by the malaria parasite Plasmodium gallinaceum. The experimental findings point to the feasibility of using the morphological changes on the surface of the malaria infected avian RBC (miaRBCs) as biomarkers for diagnosis. A glass substrate with a controlled surface roughness was used as part of a polydimethylsiloxane (PDMS) microfluidic channels. When whole-blood samples were introduced into the channels, the miaRBCs would be preferentially slowed and eventually become immobilized on the roughened surface. The surface lesions and furrow-like structures on the miaRBC surfaces offered a markedly higher probability to interact with the roughened substrate and allowed the cells to become imobilized on the surface. The captured miaRBCs were from blood samples at various degrees of infection at 3.2%, 3.9%, 9.1%, 13.4%, 20.1%, 28%, and 37%. It was observed that the miaRBCs could be selectively captured under a wall shear rate between 2.1 to 3.2 s(-1), which was directly proportional to the flow rate through the channels. This capture rate could be improved by increasing the channel length and finer flow control. It was also found that a roughened glass substrate with ten-point-height larger than the depth of surface lesions and furrow-like structures of miaRBCs showed a substantial enhancement on the number of immobilized infected RBCs. These findings indicated that surface morphologies, including surface lesions and furrow-like structures, can serve as an alternative biomarker for malaria diagnosis.