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.

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.

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.

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.

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.

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.

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.

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.

Mosquito age and avian malaria infection.

BACKGROUND: The immune system of many insects wanes dramatically with age, leading to the general prediction that older insects should be more susceptible to infection than their younger counterparts. This prediction is however challenged by numerous studies showing that older insects are more resistant to a range of pathogens. The effect of age on susceptibility to infections is particularly relevant for mosquitoes given their role as vectors of malaria and other diseases. Despite this, the effect of mosquito age on Plasmodium susceptibility has been rarely explored, either experimentally or theoretically. METHODS: Experiments were carried out using the avian malaria parasite Plasmodium relictum and its natural vector in the field, the mosquito Culex pipiens. Both innate immune responses (number and type of circulating haemocytes) and Plasmodium susceptibility (prevalence and burden) were quantified in seven- and 17-day old females. Whether immunity or Plasmodium susceptibility are modulated by the previous blood feeding history of the mosquito was also investigated. To ensure repeatability, two different experimental blocks were carried out several weeks apart. RESULTS: Haemocyte numbers decrease drastically as the mosquitoes age. Despite this, older mosquitoes are significantly more resistant to a Plasmodium infection than their younger counterparts. Crucially, however, the age effect is entirely reversed when old mosquitoes have taken one previous non-infected blood meal. CONCLUSIONS: The results agree with previous studies showing that older insects are often more resistant to infections than younger ones. These results suggest that structural and functional alterations in mosquito physiology with age may be more important than immunity in determining the probability of a Plasmodium infection in old mosquitoes. Possible explanations for why the effect is reversed in blood-fed mosquitoes are discussed. The reversal of the age effect in blood fed mosquitoes implies that age is unlikely to have a significant impact on mosquito susceptibility in the field.

Plasmodium infection brings forward mosquito oviposition.

Invertebrate hosts often bring forward their reproductive effort in response to a parasitic infection. This is widely interpreted as a host-driven response aimed at compensating for the expected losses in future fitness as a result of parasitism. Here we report that mosquitoes bring forward their oviposition schedule when they are infected with Plasmodium, a parasite known to severely curtail mosquito fecundity. This response could aim at compensating for a negative time-dependent effect of the parasite on mosquito fitness, as infected mosquitoes seem to display a strong and progressive decrease in the quality of the eggs they lay. In addition, we show that this shift in oviposition date is dependent on mosquito strain: a comparison of several isogenic mosquitoes strains, one insecticide-susceptible and two insecticide-resistant ones, reveals that only the former shift their oviposition strategy when infected. This pattern suggests the existence of a costly host-driven response to parasitism, as insecticide-resistant mosquitoes have been shown to be in generally poorer condition.

Isospora serinuse n. sp. (Apicomplexa: Eimeriidae) from a domestic canary (Serinus canaria forma domestica) (Passeriformes: Fringillidae) in Western Australia.

A new species, Isospora serinuse n. sp., (Apicomplexa:Eimeriidae) is described from a single domestic canary (Serinus canaria forma domestica) (subspecies S. c. domestica) in Western Australia. Sporulated oocysts of Isospora serinuse n. sp. are spherical or subspherical, 25.5 (24.4-27.0) × 23.5 (22.0-24.8) µm, with a shape index (length/width) of 1.09; and a smooth bilayered oocyst wall, 1.2 µm thick (outer layer 0.9 µm, inner 0.3 µm). A polar granule is present, but a micropyle and oocyst residuum are absent. The sporocysts are lemon-shaped, 18.9 (17.8-20.2) × 11.8 (10.6-13.0) µm, with a shape index of 1.6. Stieda and substieda bodies are present, the Stieda body being a small crescent shape and the substieda being indistinct. Each sporocyst with four vermiform sporozoites arranged head to tail. A sporocyst residuum is present and composed of numerous granules of different sizes that are scattered among the sporozoites. Morphologically, the oocysts of Isospora serinuse n. sp. were different from those of all known valid Isospora spp. Molecular analysis was conducted at 3 loci: the 18S and 28S ribosomal RNA and two separate regions of subunit I of the mitochondrial cytochrome oxidase (COI) gene (designated COIa and COIb). At the 18S locus, Isospora serinuse n. sp. exhibited 97.5% similarity to Isospora sp. Tokyo from a domestic pigeon (Columba livia domestica) in Japan. At the 28S locus, I. serinuse n. sp. exhibited 94.9% similarity to Isospora anthochaerae n. sp. from a red wattlebird (Anthochaera carunculata) in Australia. At the COIa locus, I. serinuse n. sp. exhibited 95.7% similarity to Isospora sospora sp. ex Apodemus flavicollis from a yellow-necked mouse and Isospora gryphoni from an American goldfinch (Carduelis tristis) respectively. At the COIb locus, I. serinuse n. sp. exhibited 96.7% similarity to an Isospora (iSAT4) from a European pied flycatcher (Ficedula hypoleuca). Based on morphological and molecular data, this isolate is a new species of Isospora, which is named Isospora serinuse n. sp. after its host, the domestic canary (S. canaria forma domestica).

Plasmodium spp.: an experimental study on vertebrate host susceptibility to avian malaria.

The interest in experimental studies on avian malaria caused by Plasmodium species has increased recently due to the need of direct information about host-parasite interactions. Numerous important issues (host susceptibility, development of infection, the resistance and tolerance to avian malaria) can be answered using experimental infections. However, specificity of genetically different lineages of malaria parasites and their isolates is largely unknown. This study reviews recent experimental studies and offers additional data about susceptibility of birds to several widespread cytochrome b (cyt b) lineages of Plasmodium species belonging to four subgenera. We exposed two domesticated avian hosts (canaries Serinus canaria and ducklings Anas platyrhynchos) and also 16 species of common wild European birds to malaria infections by intramuscular injection of infected blood and then tested them by microscopic examination and PCR-based methods. Our study confirms former field and experimental observations about low specificity and wide host-range of Plasmodium relictum (lineages SGS1 and GRW11) and P. circumflexum (lineage TURDUS1) belonging to the subgenera Haemamoeba and Giovannolaia, respectively. However, the specificity of different lineages and isolates of the same parasite lineage differed between species of exposed hosts. Several tested Novyella lineages were species specific, with a few cases of successful development in experimentally exposed birds. The majority of reported cases of mortality and high parasitaemia were observed during parasite co-infections. Canaries were susceptible mainly for the species of Haemamoeba and Giovannolaia, but were refractory to the majority of Novyella isolates. Ducklings were susceptible to three malaria infections (SGS1, TURDUS1 and COLL4), but parasitaemia was light (<0.01%) and transient in all exposed birds. This study provides novel information about susceptibility of avian hosts to a wide array of malaria parasite lineages, outlining directions for future experimental research on various aspects of biology and epidemiology of avian malaria.

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.

Malaria infection increases bird attractiveness to uninfected mosquitoes.

The epidemiology of vector-borne pathogens is largely determined by the host-choice behaviour of their vectors. Here, we investigate whether a Plasmodium infection renders the host more attractive to host-seeking mosquitoes. For this purpose, we work on a novel experimental system: the avian malaria parasite Plasmodium relictum, and its natural vector, the mosquito Culex pipiens. We provide uninfected mosquitoes with a choice between an uninfected bird and a bird undergoing either an acute or a chronic Plasmodium infection. Mosquito choice is assessed by microsatellite typing of the ingested blood. We show that chronically infected birds attract significantly more vectors than either uninfected or acutely infected birds. Our results suggest that malaria parasites manipulate the behaviour of uninfected vectors to increase their transmission. We discuss the underlying mechanisms driving this behavioural manipulation, as well as the broader implications of these effects for the epidemiology of malaria.

Food availability and competition do not modulate the costs of Plasmodium infection in dominant male canaries.

Understanding the different factors that may influence parasite virulence is of fundamental interest to ecologists and evolutionary biologists. It has recently been demonstrated that parasite virulence may occur partly through manipulation of host competitive ability. Differences in competitive ability associated with the social status (dominant or subordinate) of a host may determine the extent of this competition-mediated parasite virulence. We proposed that differences between subordinate and dominant birds in the physiological costs of infection may change depending on the level of competition in social groups. We observed flocks of domestic canaries to determine dominant or subordinate birds, and modified competition by providing restricted (high competition) or ad libitum food (low competition). Entire flocks were then infected with either the avian malaria parasite, Plasmodium relictum or a control. Contrary to our predictions we found that the level of competition had no effect on the outcome of infection for dominant or subordinate birds. We found that dominant birds appeared to suffer greater infection mediated morbidity in both dietary treatments, with a higher and more sustained reduction in haematocrit, and higher parasitaemia, than subordinates. Our results show that dominance status in birds can certainly alter parasite virulence, though the links between food availability, competition, nutrition and virulence are likely to be complex and multifaceted.

Isospora canaria Box, 1975 (Apicomplexa: Eimeriidae) from canaries Serinus canaria Linnaeus (Passeriformes: Fringillidae) in Brazil.

Isospora canaria Box, 1975 (Protozoa, Apicomplexa, Eimeriidae) is reported and described from canaries Serinus canaria (Linnaeus) in southeast Brazil. Its oöcysts are subspheroidal to ellipsoidal, 24.4 × 22.2 µm, with smooth, bilayered wall, ~1.2 µm. The micropyle and the oöcyst residuum are absent, but a polar granule is present. The sporocysts are lemon-shaped, 17.6 × 10.6 µm. The Stieda body is nipple-like, and substieda body is prominent and homogeneous. The sporocyst residuum is composed of scattered granules. The sporozoite has anterior and posterior refractile bodies and a nucleus. The report of this coccidium recovered from exotic canaries in South America is relevant to native passerines, mainly to Carduelis yarrellii, which are listed as vulnerable species by IUCN (International Union for Conservation of Nature and Natural Resources).

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.

Chlamydia psittaci infection in canaries heavily infested by Dermanyssus gallinae.

Dermanyssus gallinae is a haematophagous ectoparasite responsible for anemia, weight loss, dermatitis and a decrease in egg production. Dermanyssus gallinae may play a role in the modulation of the host immune system, maybe predisposing the host to some bacterial infections such as chlamydiosis. This is an important zoonosis. Humans are exposed to Chlamydia psittaci through inhalation of the agent dispersed from the infected birds. In this study, a syndrome observed in an aviary of canaries was investigated. A heavy infestation by D. gallinae was reported. Simultaneously, a C. psittaci infection was molecularly confirmed in the canaries. Combined therapy was applied successfully. The association of C. psittaci with the examined mites has been confirmed. Therefore, we think that D. gallinae have played a role in the spreading of C. psittaci infection among the canaries. Moreover, D. gallinae could have played an important role predisposing the canaries to the development of chlamydiosis, by inducing anemia and debilitation. The control of mites in the aviaries may represent a crucial step for the prevention of important infection such as chlamydiosis in birds and humans.

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.