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.

A survey of parasitic infections in Psittaciformes and Passeriformes in Mashhad, Iran.

The health, growth and fertility of avian species can be negatively affected by parasite infection. This survey assesses the presence, variety and distribution of internal and external parasites among parrots and perching birds in Mashhad, Iran. This study examined 751 caged pet birds from different species and regions in Mashhad for faecal samples and 132 oral swabs for digestive tract parasites. Furthermore, skin scrapings were conducted on 14 canaries displaying dishevelled feathers. During the study, mortalities and moribund birds that had been referred underwent necropsies to examine internal parasites. Following the formol ether faecal examination, only one Malango parrot tested positive for Heterakoidea eggs among 751 faecal samples (0.13%). Further, one cockatiel showed evidence of parasitic infection with Eimeria spp. (0.13%). However, neither Cryptosporidium nor Giardia protozoa were detected in the samples (0%). Oral swabs revealed no evidence of Trichomonas (0%). On the other hand, skin scraping revealed the presence of the mite Dermanyssus in 7 out of 14 canaries with dishevelled feathers (50%). Of 25 moribund and weak budgerigars, 2 were infected with Acuaria in their proventriculus (8%). In addition, 3 out of 14 deceased myna birds carried the nematode Diplotriana in their coelomic cavities (21.42%). In conclusion, the rate of internal parasites has been relatively low in ornamental birds of Mashhad, whereas the prevalence of external parasites has been higher.

Integrative taxonomy for the traditional coccidians (Chromista: Miozoa: Eimeriidae) from island canaries (Aves: Passeriformes: Fringillidae): Worldwide distribution, morphological and molecular characterization, revaluations and establishment of junior synonyms.

Island canaries Serinus canaria (Linnaeus) are finches native to the North Atlantic Islands, however, they have a worldwide distribution in captivity due to their relevance as a pet bird. Coccidians are the most reported parasites of passerines worldwide, both in the wild and in captivity, being frequently associated with disease in passerines kept in rehabilitation centers and commercial breeders. This study aimed to identify coccidians from island canaries kept in captivity in Brazil. Three hundred and fifteen genomic DNA extracted from fecal samples of island canaries from different breeders from Southern and Southeastern Brazil were used to perform a nested PCR assay to amplify a partial fragment of the 28S small subunit ribosomal RNA gene (28S) of Isospora spp. Microscopic screening and morphological identification of Isospora oocysts was performed in fecal samples corresponding to PCR positive DNA samples. Fecal samples have been formalin-stored for approximately four years. Positivity rate for both microscopy and PCR was 10.5% (33/315). Posteriorly, Isospora serini (Aragão, 1933) Box, 1975 and Isospora canaria Box, 1975 were morphologically identified from fresh fecal samples of island canaries maintained by a breeder in the State of São Paulo, Southeastern Brazil, providing a genotypic characterization via sequencing of the mitochondrial cytochrome c oxidase subunit 1 (COI) and 28S genes. The 28S and COI sequences referring to the morphological identification of I. canaria was, respectively, 100% and 99% similar to sequences deposited as Isospora serinuse Yang, Brice, Elliot & Ryan, 2015 from island canaries kept in a rehabilitation center in Australia. The COI sequence referring to the morphological identification of I. serini was 100% similar to a sequence of an extraintestinal Isospora, corroborating this identification/sequencing since I. serini is the first isosporan with an extra-intestinal cycle demonstrated. The comparison of morphological and molecular data from I. canaria and I. serini from this study with published data of Isospora spp. from canaries worldwide, allowed the specific identification from preliminary generic identifications, correction of misidentifications, as well as the establishment of junior synonyms. Finally, this study provides morphological and molecular data that ensure the correct identification of the two Isospora spp. from island canaries in future studies worldwide.

Culex quinquefasciatus (Diptera: Culicidae) survivorship following the ingestion of bird blood infected with Haemoproteus sp. parasites.

Arthropod vectors are frequently exposed to a diverse assemblage of parasites, but the consequence of these infections on their biology and behavior are poorly understood. We experimentally evaluated whether the ingestion of a common protozoan parasite of avian hosts (Haemoproteus spp.; Haemosporida: Haemoproteidae) impacted the survivorship of Culex quinquefasciatus (Say) (Diptera: Culicidae). Blood was collected from wild northern cardinals (Cardinalis cardinalis) in College Station, Texas, and screened for the presence of Haemoproteus spp. parasites using microscopic and molecular methods. Experimental groups of Cx. quinquefasciatus mosquitoes were offered Haemoproteus-positive cardinal blood through an artificial feeding apparatus, while control groups received Haemoproteus-negative cardinal blood or domestic canary (Serinus canaria domestica) blood. Culex quinquefasciatus mosquitoes exposed to Haemoproteus infected cardinal blood survived significantly fewer days than mosquitoes that ingested Haemoproteus-negative cardinal blood. The survival of mosquitoes fed on positive cardinal blood had a median survival time of 18 days post-exposure and the survival of mosquitoes fed on negative cardinal blood exceeded 50% across the 30 day observation period. Additionally, mosquitoes that fed on canary controls survived significantly fewer days than cardinal negative controls, with canary control mosquitoes having a median survival time of 17 days. This study further supports prior observations that Haemoproteus parasites can be pathogenic to bird-biting mosquitoes, and suggests that Haemoproteus parasites may indirectly suppress the transmission of co-circulating vector-borne pathogens by modulating vector survivorship. Our results also suggest that even in the absence of parasite infection, bloodmeals from different bird species can influence mosquito survivorship.

Different distribution of malaria parasite in left and right extremities of vertebrate hosts translates into differences in parasite transmission.

Malaria, a vector-borne disease caused by Plasmodium spp., remains a major global cause of mortality. Optimization of disease control strategies requires a thorough understanding of the processes underlying parasite transmission. While the number of transmissible stages (gametocytes) of Plasmodium in blood is frequently used as an indicator of host-to-mosquito transmission potential, this relationship is not always clear. Significant effort has been made in developing molecular tools that improve gametocyte density estimation and therefore prediction of mosquito infection rates. However a significant level of uncertainty around estimates remains. The weakness in the relationship between gametocyte burden, measured from a blood sample, and the mosquito infection rate could be explained by a non-homogeneous distribution of gametocytes in the bloodstream. The estimated gametocyte density would then only be a single snapshot that does not reflect the host infectivity. This aspect of Plasmodium infection, however, remains largely neglected. In both humans and birds, we found here that the gametocyte densities differed depending on which side of the body the sample was taken, suggesting that gametocytes are not homogeneously distributed within the vertebrate host. We observed a fluctuating asymmetry, in other words, the extremity of the body with the highest density of parasites is not always the same from one individual to another. An estimation of gametocyte density from only one blood sample, as is commonly measured, could, therefore, over- or underestimated the infectivity of gametocyte carriers. This might have important consequences on the epidemiology of the disease since we show that this variation influences host-to-mosquito transmission. Vectors fed on the least infected body part had a lower parasite burden than those fed on the most infected part. The heterogeneous distribution of gametocytes in bloodstream should be considered to improve diagnosis and test new malaria control strategies.

Parasitological and molecular survey of scattered parasitism by trichomonads in some avian species in Iran.

Outbreaks of avian trichomonosis are being reported worldwide; meanwhile, the genetic and virulence variations are under investigation. In this study, the occurrence and genetic variability of oral or faecal trichomonads among various avian species were investigated. Samples obtained from either the oropharyngeal cavity, crop/oesophagus, droppings/cloaca, or conjunctival swabs of avian species were inspected for flagellates. Phylogenetic analysis of partial ITS1-5.8s rRNA-ITS2 sequences from selected samples was performed to investigate the genetic diversity of the isolates. Investigation of 737 birds revealed an infection rate of 15.7% in the upper gastrointestinal tract, 7.3% in the faecal samples, and 0.7% involvement of the conjunctiva. Phylogenetic analysis of partial ITS1-5.8s rRNA-ITS2 sequences from selected samples, identified genotypes A and B of Trichomonas gallinae and genogroups A-C and E of Tetratrichomonas gallinarum. A novel ITS genotype of intestinal trichomonads was also detected in hooded crow (Corvus cornix) and common mynah (Acridotheres tristis). In the present study, in addition to Columbiformes and Falconiformes, trichomonads were detected in Passeriformes and Galliformes with the involvement of organs other than the gastrointestinal tract. Genotype A T. gallinae was detected in domestic pigeons (Columba livia domestica), a laughing dove (Spilopelia senegalensis), a common kestrel (Falco tinnunculus), a budgerigar (Melopsittacus undulates), and a canary (Serinus canaria). Distinct genotype B was detected in a common mynah and a budgerigar. Genogroups A-C of T. gallinarum were also demonstrated in Galliformes and Anseriformes. Furthermore, two novel trichomonad ITS genotypes were detected in hooded crows and a common mynah warranting detailed multi-locus molecular analysis.RESEARCH HIGHLIGHTSITS diversity of trichomonads was shown in various avian species.Diversity of the parasites' target organ and clinical manifestations was demonstrated.Two novel ITS genotype trichomonads from common mynah and hooded crow were identified.

Different paths - the same virulence: experimental study on avian single and co-infections with Plasmodium relictum and Plasmodium elongatum.

Co-infections are prevalent worldwide, however, we are still struggling to understand interactions between different parasites and their impacts on host fitness. In the present experimental study we analysed the infection dynamics of two avian malarial parasites Plasmodium elongatum (genetic lineage pERIRUB01) and Plasmodium relictum (genetic lineage pSGS1) and their impacts on host health during single and co-infections. We reveal that P. elongatum intensity of parasitemia is enhanced by the presence of P. relictum during co-infection, while the parasitemia of P. relictum stays the same. This illustrates how development of a parasite (P. elongatum) which infects both mature and young (polychromatic) red blood cells (RBCs) is facilitated during co-infection with a parasite which specialises in adult RBCs only (P. relictum). The virulence of co-infections was similar to that of the more virulent parasite (P. elongatum). However, the profile of infection and the mechanisms that caused mortality were different. Birds infected with P. elongatum only start to die due to non-regenerative anaemia, when intensity of parasitemia is light and the number of polychromatic RBCs decrease dramatically. Meanwhile, co-infected birds start to die when the mean intensity of parasitemia reaches 10% and the number of polychromatic RBCs increases abnormally, reflecting regenerative anaemia. Our findings reveal that typically measured parameters of virulence (e.g., mortality rate, level of hematocrit) can be the same during single and co-infections, but the mechanisms responsible for the observed virulence can be different. This information serves a better understanding of the processes underpinning the interactions of co-infected parasite species.

The effect of dietary antioxidant supplementation in a vertebrate host on the infection dynamics and transmission of avian malaria to the vector.

Host susceptibility to parasites is likely to be influenced by intrinsic factors, such as host oxidative status determined by the balance between pro-oxidant production and antioxidant defences. As a result, host oxidative status acts as an environmental factor for parasites and may constrain parasite development. We evaluated the role of host oxidative status on infection dynamics of an avian malarial parasite by providing canaries (Serinus canaria) with an antioxidant supplementation composed of vitamin E (a lipophilic antioxidant) and olive oil, a source of monounsaturated fatty acids. Another group received a standard, non-supplemented food. Half of the birds in each group where then infected with the haemosporidian parasite, Plasmodium relictum. We monitored the parasitaemia, haematocrit level, and red cell membrane resistance, as well as the transmission success of the parasite to its mosquito vector, Culex pipiens. During the acute phase, the negative effect of the infection was more severe in the supplemented group, as shown by a lower haematocrit level. Parasitaemia was lower in the supplemented group during the chronic phase only. Mosquitoes fed on supplemented hosts were more often infected than mosquitoes fed on the control group. These results suggest that dietary antioxidant supplementation conferred protection against Plasmodium in the long term, at the expense of a short-term negative effect. Malaria parasites may take advantage of antioxidants, as shown by the increased transmission rate in the supplemented group. Overall, our results suggest an important role of oxidative status in infection outcome and parasite transmission.

Avian malaria co-infections confound infectivity and vector competence assays of Plasmodium homopolare.

Currently, there are very few studies of avian malaria that investigate relationships among the host-vector-parasite triad concomitantly. In the current study, we experimentally measured the vector competence of several Culex mosquitoes for a newly described avian malaria parasite, Plasmodium homopolare. Song sparrow (Melospiza melodia) blood infected with a low P. homopolare parasitemia was inoculated into a naïve domestic canary (Serinus canaria forma domestica). Within 5 to 10 days post infection (dpi), the canary unexpectedly developed a simultaneous high parasitemic infection of Plasmodium cathemerium (Pcat6) and a low parasitemic infection of P. homopolare, both of which were detected in blood smears. During this infection period, PCR detected Pcat6, but not P. homopolare in the canary. Between 10 and 60 dpi, Pcat6 blood stages were no longer visible and PCR no longer amplified Pcat6 parasite DNA from canary blood. However, P. homopolare blood stages remained visible, albeit still at very low parasitemias, and PCR was able to amplify P. homopolare DNA. This pattern of mixed Pcat6 and P. homopolare infection was repeated in three secondary infected canaries that were injected with blood from the first infected canary. Mosquitoes that blood-fed on the secondary infected canaries developed infections with Pcat6 as well as another P. cathemerium lineage (Pcat8); none developed PCR detectable P. homopolare infections. These observations suggest that the original P. homopolare-infected songbird also had two un-detectable P. cathemerium lineages/strains. The vector and host infectivity trials in this study demonstrated that current molecular assays may significantly underreport the extent of mixed avian malaria infections in vectors and hosts.

Detection and molecular characterization of Cryptosporidium spp. in captive canaries (Serinus canaria) using different diagnostic methods.

This study used several diagnostic methods to examine the occurrence of and molecularly characterize Cryptosporidium spp. in captive canaries (Serinus canaria) in southern and southeastern Brazil. A total of 498 fecal samples were purified by centrifugal-flotation using Sheather's solution. Cryptosporidium spp. diagnosis was performed using three diagnostic methods: malachite green negative staining, nested PCR targeting the 18S rRNA gene, followed by sequencing the amplified fragments, and duplex real-time PCR targeting the 18S rRNA specific to detect Cryptosporidium galli and Cryptosporidium avian genotype III. The overall positivity for Cryptosporidium spp. (total samples positive in at least one protocol) from the microscopic analysis, nested PCR and duplex real-time PCR protocol results was 13.3% (66/498). The positivity rates were 2.0% (10/498) and 4.6% (23/498) for Cryptosporidium spp. by microscopy and nested PCR, respectively. Sequencing of 20 samples amplified by nested PCR identified C. galli (3.0%; 15/498), Cryptosporidium avian genotype I (0.8%; 4/498) and Cryptosporidium avium (0.2%; 1/498). Duplex real-time PCR revealed a positivity of 7.8% (39/498) for C. galli and 2.4% (12/498) for avian genotype III. Malachite green negative staining differed significantly from nested PCR in detecting Cryptosporidium spp. Duplex real-time PCR was more sensitive than nested PCR/sequencing for detecting gastric Cryptosporidium in canaries.

Detection and molecular characterization of Cryptosporidium spp. in captive canaries (Serinus canaria) using different diagnostic methods / Detecção e caracterização molecular de Cryptosporidium spp. em canários mantidos em cativeiro (Serinus canaria) por diferentes métodos de diagnóstico

Abstract This study used several diagnostic methods to examine the occurrence of and molecularly characterize Cryptosporidium spp. in captive canaries (Serinus canaria) in southern and southeastern Brazil. A total of 498 fecal samples were purified by centrifugal-flotation using Sheather's solution. Cryptosporidium spp. diagnosis was performed using three diagnostic methods: malachite green negative staining, nested PCR targeting the 18S rRNA gene, followed by sequencing the amplified fragments, and duplex real-time PCR targeting the 18S rRNA specific to detect Cryptosporidium galli and Cryptosporidium avian genotype III. The overall positivity for Cryptosporidium spp. (total samples positive in at least one protocol) from the microscopic analysis, nested PCR and duplex real-time PCR protocol results was 13.3% (66/498). The positivity rates were 2.0% (10/498) and 4.6% (23/498) for Cryptosporidium spp. by microscopy and nested PCR, respectively. Sequencing of 20 samples amplified by nested PCR identified C. galli (3.0%; 15/498), Cryptosporidium avian genotype I (0.8%; 4/498) and Cryptosporidium avium (0.2%; 1/498). Duplex real-time PCR revealed a positivity of 7.8% (39/498) for C. galli and 2.4% (12/498) for avian genotype III. Malachite green negative staining differed significantly from nested PCR in detecting Cryptosporidium spp. Duplex real-time PCR was more sensitive than nested PCR/sequencing for detecting gastric Cryptosporidium in canaries.

Resumo Este trabalho teve como objetivos determinar a ocorrência e realizar a caracterização molecular de Cryptosporidium spp. em 498 amostras fecais de canários (Serinus canaria) criados em cativeiro, utilizando três métodos de diagnóstico: análise microscópica pela coloração negativa com verde malaquita, nested PCR seguida de sequenciamento dos fragmentos amplificados e PCR duplex em tempo real específica para detecção de Cryptosporidium galli e Cryptosporidium genótipo III de aves. A positividade total para Cryptosporidium spp. (total de amostras positivas em pelo menos um método de diagnóstico) obtida pela análise microscópica, nested PCR e PCR duplex em tempo real foi de 13,3% (66/498). As taxas de positividade para Cryptosporidium spp. foram 2,0% (10/498) e 4,6% (23/498) por microscopia e nested PCR, respectivamente. O sequenciamento de 20 amostras amplificadas pela nested PCR identificou C. galli (3,0%; 15/498), Cryptosporidium genótipo I de aves (0,8%; 4/498) e Cryptosporidium avium (0,2%; 1/498). A PCR duplex em tempo real revelou positividade de 7,8% (39/498) para C. galli e 2,4% (12/498) para Cryptosporidium genótipo III de aves. A análise microscópica diferiu significativamente da nested PCR para detecção de Cryptosporidium spp. A PCR duplex em tempo real apresentou maior sensibilidade que a nested PCR/sequenciamento para detectar as espécies/genótipos gástricos de Cryptosporidium.

Biting midges (Ceratopogonidae) as vectors of avian trypanosomes.

BACKGROUND: Although avian trypanosomes are widespread parasites, the knowledge of their vectors is still incomplete. Despite biting midges (Diptera: Ceratopogonidae) are considered as potential vectors of avian trypanosomes, their role in transmission has not been satisfactorily elucidated. Our aim was to clarify the potential of biting midges to sustain the development of avian trypanosomes by testing their susceptibility to different strains of avian trypanosomes experimentally. Moreover, we screened biting midges for natural infections in the wild. RESULTS: Laboratory-bred biting midges Culicoides nubeculosus were highly susceptible to trypanosomes from the Trypanosoma bennetti and T. avium clades. Infection rates reached 100%, heavy infections developed in 55-87% of blood-fed females. Parasite stages from the insect gut were infective for birds. Moreover, midges could be infected after feeding on a trypanosome-positive bird. Avian trypanosomes can thus complete their cycle in birds and biting midges. Furthermore, we succeeded to find infected blood meal-free biting midges in the wild. CONCLUSIONS: Biting midges are probable vectors of avian trypanosomes belonging to T. bennetti group. Midges are highly susceptible to artificial infections, can be infected after feeding on birds, and T. bennetti-infected biting midges (Culicoides spp.) have been found in nature. Moreover, midges can be used as model hosts producing metacyclic avian trypanosome stages infective for avian hosts.

Occurrence of Ornithonyssus sylviarum in pet birds from the district of Setúbal, Portugal.

Ornithonyssus sylviarum is a blood-feeding ectoparasite of birds and the most serious pest in poultry farms in North America. Although the mites are typically adapted to temperate climates, information on this mite in Europe is sparse, and Dermanyssus gallinae is considered to be the only mite impacting the poultry industry. The present study reports the occurrence of O. sylviarum in pet birds in Portugal. Mites were collected directly from birds and with traps placed in cages and nests at 20 different sampling places belonging to 6 municipalities in the district of Setúbal. In a total of 217 birds, O. sylviarum was identified in 47 out of 147 (32.0%) canaries (Serinus canaria), 14 out of 21 (14.3%) estrildid finches, 1 out of 24 (4.2%) budgerigars (Melopsittacus undulatus) and 1 out of 15 (6.7%) lovebirds (Agapornis spp.). Mites of the genus Dermanyssus were identified in 8 canaries (5.4%), 8 estrildid finches (38.1%) and 1 lovebird (6.7%). No mites were found in 6 cockatiels (Nymphicus hollandicus), 2 African grey parrots (Psittacus erithacus), 1 Bourke's parrot (Neophema bourkii) and 1 rose-ringed parakeet (Psittacula krameri). Considering the zoonotic potential and the risk of dissemination to poultry, the present findings underline the need for further monitoring of O. sylviarum in the wild and domestic avifauna in Portugal.

Trichomonosis outbreak in a flock of canaries (Serinus canaria f. domestica) caused by a finch epidemic strain of Trichomonas gallinae.

In the present paper, an outbreak of trichomonosis in a flock of 15 breeding pairs of canaries is described. Trichomonosis was diagnosed on characteristic clinical signs, microscopic examination of crop/esophageal swabs, gross pathology and histopathology. Trichomonads were successfully grown in culture media and were characterized by multi-locus sequence typing. The three genomic loci ITS1-5.8S-ITS2, 18S rRNA and Fe-hydrogenase were analyzed. Molecular characterization confirmed the finch trichomonosis strain, identical to the strain that caused emerging disease in free-living passerine birds in Europe. Flock treatment with metronidazole (200mg/L) in drinking water for 5days was partially effective. After individual treatment with oral application of metronidazole (20mg/kg SID) for 5days no further clinical signs were observed in the flock over next 30 months.

Characterization of two complete Isospora mitochondrial genomes from passerine birds: Isospora serinuse in a domestic canary and Isospora manorinae in a yellow-throated miner.

The genus term Isospora is now applied specifically to parasites of birds, with the term Cystoisospora preferred for parasites which infect mammals. Isospora is a common parasitic coccidian in birds worldwide, especially in passerine birds, in which it can cause systemic coccidiosis. The complete mitochondrial genome sequences from two recently identified Isospora species; Isospora serinuse in a domestic canary and Isospora manorinae in a yellow-throated miner, were sequenced and compared with those of other closely related coccidian species. The complete mitochondrial genome sequence for Isospora serinuse is 6260bp in size and 6223bp for Isospora manorinae. The mitochondrial genomes of Isospora serinuse and Isospora manorinae include three protein-coding genes (COI, COIII and CytB), 19 LSU and 14 SSU rDNA fragments, including one newly identified putative LSU fragment in Isospora sp. The arrangement of coding regions in these two Isospora species were identical to that of available Isospora sp. and Eimeria spp. mitochondrial genomes and the start codon usage for protein coding genes was conservative. Phylogenetic analysis of the mt genome of the two Isospora species based on the three coding regions further support that the monophyletic nature of avian Isospora.

Exposure of the mosquito vector Culex pipiens to the malaria parasite Plasmodium relictum: effect of infected blood intake on immune and antioxidant defences, fecundity and survival.

BACKGROUND: The intake of a Plasmodium-infected blood meal may affect mosquito physiology and a series of trade-offs may occur, in particular between immune defences, reproduction and self-maintenance. We evaluated the cost of exposure to Plasmodium in the mosquito vector by investigating the effect of exposure on fecundity and survival and the implication of immune and antioxidant defences in mediating this cost. METHODS: We used the natural Culex pipiens-Plasmodium relictum association. We exposed female mosquitoes to increasing levels of parasites by allowing them to feed either on uninfected canaries, Serinus canaria, (unexposed mosquitoes) or on infected canaries with low (low exposure) or high (high exposure) parasitaemia. We recorded blood meal size, fecundity (laying probability and clutch size) and survival. We quantified the expression of genes involved in immune and antioxidant defences (nitric oxide synthase, NOS; superoxide dismutase, SOD; glucose-6-phosphate dehydrogenase, G6PDH). RESULTS: We found that the laying probability of exposed females decreased with increasing exposure to the parasite and with increasing SOD expression. Clutch size of exposed females was higher compared to unexposed ones for similar blood meal size and was positively correlated to the NOS expression. We found no effect of exposure on survival. After blood meal intake, SOD increased in the three groups, NOS increased in exposed females and G6PDH increased in highly exposed females only. CONCLUSIONS: Our results illustrated a trade-off between fight against the parasite and reproduction and a cost of exposure which might be mediated by the investment in immune and/or antioxidant defences. They also showed that this trade-off could lead to opposed outcome, potentially depending on the vector physiological status. Finally, they highlighted that the ingestion of a Plasmodium-infected blood meal may affect mosquito life history traits in a complex way.

The Strategy to Survive Primary Malaria Infection: An Experimental Study on Behavioural Changes in Parasitized Birds.

Avian malaria parasites (Haemosporida, Plasmodium) are of cosmopolitan distribution, and they have a significant impact on vertebrate host fitness. Experimental studies show that high parasitemia often develops during primary malaria infections. However, field studies only occasionally reveal high parasitemia in free-living birds sampled using the traditional methods of mist-netting or trapping, and light chronic infections predominate. The reason for this discrepancy between field observation and experimental data remains insufficiently understood. Since mist-netting is a passive capture method, two main parameters determine its success in sampling infected birds in wildlife, i. e. the presence of parasitized birds at a study site and their mobility. In other words, the trapping probability depends on the survival rate of birds and their locomotor activity during infection. Here we test (1) the mortality rate of wild birds infected with Plasmodium relictum (the lineage pSGS1), (2) the changes in their behaviour during presence of an aerial predator, and (3) the changes in their locomotor activity at the stage of high primary parasitemia.We show that some behavioural features which might affect a bird's survival during a predator attack (time of reaction, speed of flush flight and take off angle) did not change significantly during primary infection. However, the locomotor activity of infected birds was almost halved compared to control (non-infected) birds during the peak of parasitemia. We report (1) the markedly reduced mobility and (2) the 20% mortality rate caused by P. relictum and conclude that these factors are responsible for the underrepresentation of birds in mist nets and traps during the stage of high primary parasitemia in wildlife. This study indicates that the widespread parasite, P. relictum (pSGS1) influences the behaviour of birds during primary parasitemia. Experimental studies combined with field observations are needed to better understand the mechanisms of pathogenicity of avian malaria parasites and their influence on bird populations.

Description, molecular characterisation, diagnostics and life cycle of Plasmodium elongatum (lineage pERIRUB01), the virulent avian malaria parasite.

Plasmodium elongatum causes severe avian malaria and is distributed worldwide. This parasite is of particular importance due to its ability to develop and cause lethal malaria not only in natural hosts, but also in non-adapted endemic birds such as the brown kiwi and different species of penguins. Information on vectors of this infection is available but is contradictory. PCR-based analysis indicated the possible existence of a cluster of closely related P. elongatum lineages which might differ in their ability to develop in certain mosquitoes and birds. This experimental study provides information about molecular and morphological characterisation of a virulent P. elongatum strain (lineage pERIRUB01) isolated from a naturally infected European robin, Erithacus rubecula. Phylogenetic analysis based on partial cytochrome b gene sequences showed that this parasite lineage is closely related to P. elongatum (lineage pGRW6). Blood stages of both parasite lineages are indistinguishable, indicating that they belong to the same species. Both pathogens develop in experimentally infected canaries, Serinus canaria, causing death of the hosts. In both these lineages, trophozoites and erythrocytic meronts develop in polychromatic erythrocytes and erythroblasts, gametocytes parasitize mature erythrocytes, exoerythrocytic stages develop in cells of the erythrocytic series in bone marrow and are occasionally reported in spleen and liver. Massive infestation of bone marrow cells is the main reason for bird mortality. We report here on syncytium-like remnants of tissue meronts, which slip out of the bone marrow into the peripheral circulation, providing evidence that the syncytia can be a template for PCR amplification. This finding contributes to better understanding positive PCR amplifications in birds when parasitemia is invisible and improved diagnostics of abortive haemosporidian infections. Sporogony of P. elongatum (pERIRUB01) completes the cycle and sporozoites develop in widespread Culex quinquefasciatus and Culex pipiens pipiens form molestus mosquitoes. This experimental study provides information on virulence and within species lineage diversity in a single pathogenic species of haemosporidian parasite.

Plasmodium delichoni n. sp.: description, molecular characterisation and remarks on the exoerythrocytic merogony, persistence, vectors and transmission.

Malaria parasite Plasmodium (Novyella) delichoni n. sp. (Haemosporida, Plasmodiidae) was found in a widespread Eurasian songbird, the common house martin Delichon urbicum (Hirundinidae). It is described based on the morphology of its blood stages and segments of the mitochondrial cytochrome b and apicoplast genes, which can be used for molecular identification of this species. Erythrocytic meronts and gametocytes are strictly nucleophilic, and mature gametocytes possess pigment granules of markedly variable size, including large ones (1 µm in length). Due to these features, P. delichoni can be readily distinguished from all described species of avian malaria parasites belonging to subgenus Novyella. Additionally, mature erythrocytic merozoites contain a dense clump of chromatin, a rare character in avian malaria parasites. Erythrocytic merogony is asynchronous. Illustrations of blood stages of the new species are given, and phylogenetic analysis identifies DNA lineages closely related to this parasite. Domestic canary Serinus canaria and Eurasian siskin Carduelis spinus were infected after subinoculation of infected blood obtained from the house martin. Parasitemia was long lasting in both these hosts, but it was high (up to 70 %) in Eurasian siskins and low (up to 1 %) in canaries. Mortality was not observed, and histological examination and chromogenic in situ hybridisation did not reveal secondary exoerythrocytic meronts (phanerozoites) in the exposed birds. It is likely that persistence of this infection occurs due to long-lasting parasitemia in avian hosts. Sporogony was abortive in mosquitoes Culex pipiens pipiens form molestus, Culex quinquefasciatus and Aedes aegypti at gametogenesis or ookinete stages. The new species is absent from juvenile birds at breeding sites in Europe, indicating that transmission occurs at African wintering grounds.

A method to preserve low parasitaemia Plasmodium-infected avian blood for host and vector infectivity assays.

BACKGROUND: Avian malaria vector competence studies are needed to understand more succinctly complex avian parasite-vector-relations. The lack of vector competence trials may be attributed to the difficulty of obtaining gametocytes for the majority of Plasmodium species and lineages. To conduct avian malaria infectivity assays for those Plasmodium spp. and lineages that are refractory to in vitro cultivation, it is necessary to obtain and preserve for short periods sufficient viable merozoites to infect naïve donor birds to be used as gametocyte donors to infect mosquitoes. Currently, there is only one described method for long-term storage of Plasmodium spp.-infected wild avian blood and it is reliable at a parasitaemia of at least 1%. However, most naturally infected wild-caught birds have a parasitaemia of much less that 1%. To address this problem, a method for short-term storage of infected wild avian blood with low parasitaemia (even ≤ 0.0005%) has been explored and validated. METHODS: To obtain viable infective merozoites, blood was collected from wild birds using a syringe containing the anticoagulant and the red blood cell preservative citrate phosphate dextrose adenine solution (CPDA). Each blood sample was stored at 4 °C for up to 48 h providing sufficient time to determine the species and parasitaemia of Plasmodium spp. in the blood by morphological examination before injecting into donor canaries. Plasmodium spp.--infected blood was inoculated intravenously into canaries and once infection was established, Culex stigmatosoma, Cx. pipiens and Cx. quinquefasciatus mosquitoes were then allowed to feed on the infected canaries to validate the efficacy of this method for mosquito vector competence assays. RESULTS: Storage of Plasmodium spp.--infected donor blood at 4 °C yielded viable parasites for 48 h. All five experimentally-infected canaries developed clinical signs and were infectious. Pathologic examination of three canaries that later died revealed splenic lesions typical of avian malaria infection. Mosquito infectivity assays demonstrated that Cx. stigmatosoma and Cx. pipiens were competent vectors for Plasmodium cathemerium. CONCLUSIONS: A simple method of collecting and preserving avian whole blood with malaria parasites of low parasitaemia (≤ 0.0005%) was developed that remained viable for further experimental bird and mosquito infectivity assays. This method allows researchers interested in conducting infectivity assays on target Plasmodium spp. to collect these parasites directly from nature with minimal impact on wild birds.