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Abstract Background Blood parasites have been studied intensely in many families of avian hosts, but corvids, a particularly cosmopolitan family, remain underexplored. Haemosporidian parasites of the common raven (Corvus corax) have not been studied, although it is the largest, most adaptable, and widespread corvid. Genetic sequence data from parasites of ravens can enhance the understanding of speciation patterns and specificity of haemosporidian parasites in corvids, and shed light how these hosts cope with parasite pressure. Methods A baited cage trap was used to catch 86 ravens and a nested PCR protocol was used to amplify a 479 bp fragment of the haemosporidian cytochrome b gene from the samples. The obtained sequences were compared with the MalAvi database of all published haemosporidian lineages and a phylogenetic tree including all detected raven parasites was constructed. An examination of blood smears was performed for assessment of infection intensity. Results Twenty blood parasite lineages were recovered from ravens caught in a wild population in Bulgaria. The prevalence of generalist Plasmodium lineages was 49%, and the prevalence of Leucocytozoon lineages was 31%. Out of 13 detected Leucocytozoon lineages six were known from different corvids, while seven others seem to be specific to ravens. A phylogenetic reconstruction suggests that Leucocytozoon lineages of ravens and other corvids are not monophyletic, with some groups appearing closely related to parasites of other host families. Conclusions Several different, morphologically cryptic groups of Leucocytozoon parasites appear to infect corvids. Ravens harbour both generalist corvid Leucocytozoon as well as apparently species-specific lineages. The extraordinary breeding ecology and scavenging lifestyle possibly allow ravens to evade vectors and have relatively low blood parasite prevalence compared to other corvids.

Avian blood parasites are remarkably diverse and frequently occur in co-infections, which predominate in wildlife. This makes wildlife pathogen research challenging, particularly if they belong to closely related groups, resulting in diagnostic problems and poor knowledge about such infections as well as the patterns of their co-occurrence and interactions. This is particularly true due to the periodicity (circadian rhythms) of parasitemia, which means that different parasitemia and parasite stages might be found throughout the day. We analysed blood samples from a Eurasian blackbird (Turdus merula) and a Song thrush (Turdus philomelos). This study aimed to describe a new avian Lankesterella species and molecularly characterize and redescribe Splendidofilaria mavis, a common avian filarioid nematode. Additionally, it was possible to investigate the circadian rhythms of the avian blood parasites belonging to Plasmodium, Haemoproteus, Leucocytozoon, and Trypanosoma, which occurred in co-infection in the same avian host individuals. Different circadian rhythms were seen in different parasites, with Plasmodium sp. peaks occurring at midday, Leucocytozoon spp. peaks mainly during the evening and night, and Trypanosoma spp. and microfilariae peaks at midnight. No periodicity was seen in Haemoproteus and Lankesterella species infections. The time of parasitemia peaks most likely coincides with the time of vectors’ activity, and this should be beneficial for transmission. Knowledge about the circadian rhythms is needed for better understanding patterns in host-parasite interactions and disease transmission.

Background: Haemosporidians (Apicomplexa, Protista) are obligate heteroxenous parasites of vertebrates and blood-sucking dipteran insects. Avian haemosporidians comprise more than 250 species traditionally classified into four genera, Plasmodium, Haemoproteus, Leucocytozoon, and Fallisia. However, analyses of the mitochondrial CytB gene revealed a vast variety of lineages not yet linked to morphospecies. This study aimed to analyse and discuss the data of haemosporidian lineages isolated from birds of the family Turdidae, to visualise host and geographic distribution using DNA haplotype networks and to suggest directions for taxonomy research on parasite species. Methods: Haemosporidian CytB sequence data from 350 thrushes were analysed for the present study and complemented with CytB data of avian haemosporidians gathered from Genbank and MalAvi database. Maximum Likelihood trees were calculated to identify clades featuring lineages isolated from Turdidae species. For each clade, DNA haplotype networks were calculated and provided with information on host and geographic distribution. Results: In species of the Turdidae, this study identified 82 Plasmodium, 37 Haemoproteus, and 119 Leucocytozoon lineages, 68, 28, and 112 of which are mainly found in this host group. Most of these lineages cluster in the clades, which are shown as DNA haplotype networks. The lineages of the Leucocytozoon clades were almost exclusively isolated from thrushes and usually were restricted to one host genus, whereas the Plasmodium and Haemoproteus networks featured multiple lineages also recovered from other passeriform and non-passeriform birds. Conclusion: This study represents the first attempt to summarise information on the haemosporidian parasite lineages of a whole bird family. The analyses allowed the identification of numerous groups of related lineages, which have not been linked to morphologically defined species yet, and they revealed several cases in which CytB lineages were probably assigned to the wrong morphospecies. These taxonomic issues are addressed by comparing distributional patterns of the CytB lineages with data from the original species descriptions and further literature. The authors also discuss the availability of sequence data and emphasise that MalAvi database should be considered an extremely valuable addition to GenBank, but not a replacement. [ABSTRACT FROM AUTHOR]

Parasites may affect the dynamics of bird populations. Plasmodium, Leucocytozoon and Haemoproteus are well-known avian haematozoa that can trigger decreased productivity and high mortality in domesticated birds. In this study, we evaluated the prevalence of avian blood parasites (Plasmodium, Leucocytozoon and Haemoproteus) against 335 birds of 8 species in the Yazd province in central Iran. To detect blood parasites, Giemsa-stained blood smears were prepared. Of the birds, 11.64% (39/335) were infected with at least one parasite genus, particularly Haemoproteus (32.6%; 23/335). The total prevalence values for Plasmodium, Haemoproteus and Leucocytozoon were 1.7, 6.8 and 2.9%, respectively. Plasmodium had lower prevalence rates of 1.7% (6/335). Among birds, pigeons, hens and ducks have the highest prevalence of Haemoproteus, Leucocytozoon and Plasmodium parasites at 1.7%, 6.8% and 2.9%, respectively. Results from this research extend our knowledge on the incidence of avian blood parasites in domesticated birds living in central Iran. The overall low incidence of avian blood parasites in birds was found in the Yazd province, Iran. [ABSTRACT FROM AUTHOR]

Identifying robust environmental predictors of infection probability is central to forecasting and mitigating the ongoing impacts of climate change on vector‐borne disease threats. We applied phylogenetic hierarchical models to a data set of 2,171 Western Palearctic individual birds from 47 species to determine how climate and landscape variation influence infection probability for three genera of haemosporidian blood parasites (Haemoproteus, Leucocytozoon, and Plasmodium). Our comparative models found compelling evidence that birds in areas with higher vegetation density (captured by the normalized difference vegetation index [NDVI]) had higher likelihoods of carrying parasite infection. Magnitudes of this relationship were remarkably similar across parasite genera considering that these parasites use different arthropod vectors and are widely presumed to be epidemiologically distinct. However, we also uncovered key differences among genera that highlighted complexities in their climate responses. In particular, prevalences of Haemoproteus and Plasmodium showed strong but contrasting relationships with winter temperatures, supporting mounting evidence that winter warming is a key environmental filter impacting the dynamics of host‐parasite interactions. Parasite phylogenetic community diversities demonstrated a clear but contrasting latitudinal gradient, with Haemoproteus diversity increasing towards the equator and Leucocytozoon diversity increasing towards the poles. Haemoproteus diversity also increased in regions with higher vegetation density, supporting our evidence that summer vegetation density is important for structuring the distributions of these parasites. Ongoing variation in winter temperatures and vegetation characteristics will probably have far‐reaching consequences for the transmission and spread of vector‐borne diseases. [ABSTRACT FROM AUTHOR]

Several factors shape lifetime reproductive success, including genetic background, body condition, environmental conditions and ecological interactions such as parasitism. Adults often show higher reproductive success than their young conspecifics, especially in long‐lived bird species, and this may be explained by the cumulative effects of an increase in reproductive experience and the selection of high‐quality individuals from one year to the next. To test whether this pattern also exists in short‐lived bird species, we used 13 years of monitoring data from two Great Tit Parus major populations. The effects of male and female age on several reproductive parameters were analysed in 419 pairs of Great Tits, while accounting for body condition and infection by haemosporidian parasites. Reproductive success was mainly affected by the age‐class of males. Pairs containing a sub‐adult male fledged one‐third fewer chicks than pairs containing an adult male. The difference was not caused by variation in male fertility but could have been caused by better parental care provided by adult birds. In addition to lower reproductive success, first‐year males also had reduced access to mating compared with adult males, suggesting an avoidance of sub‐adult males by females. Nestling body condition was positively correlated with parental body condition, and the body condition of male and female members of breeding pairs was positively correlated. Finally, the number of fledged chicks was mainly affected by the infection status of males. These results temper our previously published results showing an effect of infection on Great Tit reproduction regardless of their sex. In our previous study, and as in most cases, the status of the partner was not taken into account and we show here that this is essential because it can lead to a biased interpretation of the results. [ABSTRACT FROM AUTHOR]

Understanding the ecology and evolution of parasites is contingent on identifying the selection pressures they face across their infection landscape. Such a task is made challenging by the fact that these pressures will likely vary across time and space, as a result of seasonal and geographical differences in host susceptibility or transmission opportunities. Avian haemosporidian blood parasites are capable of infecting multiple co‐occurring hosts within their ranges, yet whether their distribution across time and space varies similarly in their different host species remains unclear. Here, we applied a new PCR method to detect avian haemosporidia (genera Haemoproteus, Leucocytozoon, and Plasmodium) and to determine parasite prevalence in two closely related and co‐occurring host species, blue tits (Cyanistes caeruleus,N = 529) and great tits (Parus major, N = 443). Our samples were collected between autumn and spring, along an elevational gradient in the French Pyrenees and over a three‐year period. Most parasites were found to infect both host species, and while these generalist parasites displayed similar elevational patterns of prevalence in the two host species, this was not always the case for seasonal prevalence patterns. For example, Leucocytozoon group A parasites showed inverse seasonal prevalence when comparing between the two host species, being highest in winter and spring in blue tits but higher in autumn in great tits. While Plasmodium relictum prevalence was overall lower in spring relative to winter or autumn in both species, spring prevalence was also lower in blue tits than in great tits. Together, these results reveal how generalist parasites can exhibit host‐specific epidemiology, which is likely to complicate predictions of host–parasite co‐evolution. [ABSTRACT FROM AUTHOR]

Hosts are typically coinfected by multiple parasite species whose interactions might be synergetic or antagonistic, producing unpredictable physiological and pathological impacts on the host. This study shows the interaction between Plasmodium spp. and Leucocytozoon spp. in birds experimentally infected or not infected with Mycoplasma gallisepticum.In 1994, the bacterium Mycoplasma gallisepticum jumped from poultry to wild birds in which it caused a major epidemic in North America. Birds infected with M. gallisepticum show conjunctivitis as well as increased levels of corticosterone.Malaria and other haemosporidia are widespread in birds, and chronic infections become apparent with the detectable presence of the parasite in peripheral blood in response to elevated levels of natural or experimental corticosterone levels.Knowing the immunosuppressive effect of corticosterone on the avian immune system, we tested the hypothesis that chronic infections of Plasmodium spp. and Leucocytozoon spp. in house finches would respond to experimental inoculation with M. gallisepticum as corticosterone levels are known to increase following inoculation.Plasmodium spp. infection intensity increased within days of M. gallisepticum inoculation as shown both by the appearance of infected erythrocytes and by the increase in the number and the intensity of positive PCR tests.Leucocytozoon spp. infection intensity increased when Plasmodium spp. infection intensity increased, but not in response to M. gallisepticum inoculation. Leucocytozoon spp. and Plasmodium spp. seemed to compete in the host as shown by a negative correlation between the changes in their PCR score when both pathogens were present in the same individual.Host responses to coinfection with multiple pathogens measured by the hematocrit and white blood cell count depended on the haemosporidian community composition. Host investment in the leukocyte response was higher in the single‐haemosporidia‐infected groups when birds were infected with M. gallisepticum.A trade‐off was observed between the immune control of the chronic infection (Plasmodium spp./Leucocytozoon spp.) and the immune response to the novel bacterial infection (M. gallisepticum). [ABSTRACT FROM AUTHOR]

The latitudinal diversity gradient (LDG) is an established macroecological pattern, but is poorly studied in microbial organisms, particularly parasites. In this study, we tested whether latitude, elevation, and host species predicted patterns of prevalence, alpha diversity, and community turnover of hemosporidian parasites. We expected parasite diversity to decrease with latitude, alongside the diversity of their hosts and vectors. Similarly, we expected infection prevalence to decrease with latitude as vector abundances decrease. Lastly, we expected parasite community turnover to increase with latitudinal distance and to be higher between rather than within host species. We tested these hypotheses by screening blood and tissue samples of three closely related avian species in a clade of North American songbirds (Turdidae: Catharus, n = 466) across 17.5° of latitude. We used a nested PCR approach to identify parasites in hemosporidian genera that are transmitted by different dipteran vectors. Then, we implemented linear‐mixed effects and generalized dissimilarity models to evaluate the effects of latitude, elevation, and host species on parasite metrics. We found high diversity of hemosporidian parasites in Catharus thrushes (n = 44 lineages) but no evidence of latitudinal gradients in alpha diversity or prevalence. Parasites in the genus Leucocytozoon were most prevalent and lineage rich in this study system; however, there was limited turnover with latitude and host species. Contrastingly, Plasmodium parasites were less prevalent and diverse than Leucocytozoon parasites, yet communities turned over at a higher rate with latitude and host species. Leucocytozoon communities were skewed by the dominance of one or two highly prevalent lineages with broad latitudinal distributions. The few studies that evaluate the hemosporidian LDG do not find consistent patterns of prevalence and diversity, which makes it challenging to predict how they will respond to global climate change. [ABSTRACT FROM AUTHOR]

Background: The behaviour of blood-sucking arthropods is a crucial determinant of blood protozoan distribution and hence of host-parasite coevolution, but it is very challenging to study in the wild. The molecular identification of parasite lineages in vectors can be a useful key to understand the behaviour and transmission patterns realised by these vectors. Methods: In this study, we collected blackflies around nests of three raptor species in the upper forest canopy in central Europe and examined the presence of vertebrate DNA and haemosporidian parasites in them. We molecularly analysed 156 blackfly individuals, their vertebrate blood meals, and the haemosporidian parasite lineages they carried. Results: We identified nine species of Simulium blackflies, largely belonging to the subgenera Nevermannia and Eusimulium. Only 1% of the collected specimens was visibly engorged, and only 4% contained remains of host DNA. However, in 29% of the blackflies Leucocytozoon lineages were identified, which is evidence of a previous blood meal on an avian host. Based on the known vertebrate hosts of the recorded Leucocytozoon lineages, we can infer that large and/or abundant birds, such as thrushes, crows, pigeons, birds of prey, owls and tits are the main targets of ornithophilic blackflies in the canopy. Blackfly species contained similar proportions of host group-specific parasite lineages and thus do not appear to be associated with particular host groups. Conclusions: The Leucocytozoon clade infecting thrushes, crows, and pigeons present in most represented blackfly species suggests a lack of association between hosts and blackflies, which can increase the probability of host switches of blood parasites. However, the composition of the simuliid species differed between nests of common buzzards, goshawks and red kites. This segregation can be explained by coinciding habitat preferences between host and vector, and may lead to the fast speciation of Leucocytozoon parasites. Thus, subtle ecological preferences and lack of host preference of vectors in the canopy may enable both parasite diversification and host switches, and enforce a habitat-dependent evolution of avian malaria parasites and related haemosporidia. [ABSTRACT FROM AUTHOR]

Haemosporidian parasites of birds are found worldwide and include the genera Haemoproteus , Plasmodium and Leucocytozoon. Infection with haemosporidian parasites can affect host physical condition and reproductive success. The aim of this study was to identify the blood parasites and parasitaemia in endemic and non-endemic passerine birds from central Mexico highlands. This study included 157 passerines representing 29 species from 17 families. Overall, 30.6% (48/157) of the birds were infected with blood parasites. Of those, Haemoproteus spp. were found in 14.0% (n = 22), Leucocytozoon spp. 12.1% (n = 19) and microfilariae 0.6% (n = 1). Blood parasites were found in 71.4% (5/7) of endemic bird species and 45.4% (10/22) of non-endemic species. Medium to high parasitaemia (number of parasites/number erythrocytes) was observed in birds with infections of Haemoproteus spp. and Leucocytozoon spp. Co-infections 3.8% (n = 6) were observed in two species of endemic birds. This study contributes to the knowledge of haemoparasites in endemic and non-endemic passerine birds from central Mexico highlands. Additional investigation on the molecular identification of haemosporidian parasites, pathogenicity and health status of these birds is necessary. Triple infection: Haemoproteus spp. (Image 2), Leucocytozoon spp. (Image 3), and microfilaria (➤) parasites (A) of a Catharus occidentalis (B). Photomicrographs. Scale-bar : 10 μm. Image 1 • This is the second recorded study of haemoparasites of endemic passerine birds from central Mexico highlands. • Of the 157 birds, 48 birds (30.6%) were infected with blood parasites. • In the birds it was identified Haemoproteus spp. 14.0%, Leucocytozoon spp. 12.1% and microfilariae 0.6%. • Medium to high parasitemia was observed of Haemoproteus spp. and Leucocytozoon spp. • Co-infections were observed in 3.8% (6/157) of all infected birds. [ABSTRACT FROM AUTHOR]

Latitudinal gradients are well-suited systems that may be helpful explaining distribution of haemosporidian parasites and host susceptibility. We studied the prevalence, diversity and drivers of haemosporidian parasites (Leucocytozoon , Plasmodium and Haemoproteus) along a latitudinal gradient (30°–56° S), that encompass the total distribution (~3,000 km) of the Thorn-tailed Rayadito (Aphrastura spinicauda) in the South American temperate forests from Chile. We analyzed 516 individuals from 18 localities between 2010 and 2017 and observed an overall prevalence of 28.3% for haemosporidian parasites. Leucocytozoon was the most prevalent parasite (25.8%). We recorded 19 distinct lineages (13 for Leucocytozoon , five for Plasmodium , and one for Haemoproteus). Differences in haemosporidian prevalence and diversity by genus and type of habitat were observed in the latitudinal gradient. Further, we support the existence of a latitudinal associate distribution of Leucocytozoids in South America, where prevalence and diversity increase toward higher latitudes. Distribution of Leucocytozoon was associated with sub-antarctic habitat (higher latitude) and explained by cold temperature and high precipitation. On the other hand, we lacked to find a latitudinal associate pattern for Plasmodium and Haemoproteus , however low prevalence and high diversity were recorded in areas considered as a hotspot of biodiversity in Central Chile. Our findings confirmed the importance of habitat and climatic variables explaining prevalence, diversity and distribution of haemosporidian parasites in a huge latitudinal gradient, belonging the distribution of the Thorn-tailed Rayadito in the world's southernmost forests ecosystems. Image 1 • Prevalence and diversity differed between haemosporidian genera and habitats in the latitudinal gradient. • Leucocytozoon was the most prevalent genus in the latitudinal distribution. • Leucocytozoon prevalence and diversity were associated to higher latitudes. • Distribution of Leucocytozoon was explained by cold temperature and high precipitation. [ABSTRACT FROM AUTHOR]

Background: Blood parasites belonging to the Apicomplexa, Trypanosomatidae and Filarioidea are widespread in birds and have been studied extensively. Microscopical examination (ME) of stained blood films remains the gold standard method for the detection of these infections in birds, particularly because co-infections predominate in wildlife. None of the available molecular tools can detect all co-infections at the same time, but ME provides opportunities for this to be achieved. However, fixation, drying and staining of blood films as well as their ME are relatively time-consuming. This limits the detection of infected hosts during fieldwork when captured animals should be released soon after sampling. It is an obstacle for quick selection of donor hosts for parasite experimental, histological and other investigations in the field. This study modified, tested and described the buffy coat method (BCM) for quick diagnostics (~ 20 min/sample) of avian blood parasites. Methods: Blood of 345 birds belonging to 42 species was collected, and each sample was examined using ME of stained blood films and the buffy coat, which was examined after centrifugation in capillary tubes and after being transferred to objective glass slides. Parasite detection using these methods was compared using sensitivity, specificity, positive and negative predictive values and Cohen's kappa index. Results: Haemoproteus, Leucocytozoon, Plasmodium, microfilariae, Trypanosoma and Lankesterella parasites were detected. BCM had a high sensitivity (> 90%) and specificity (> 90%) for detection of Haemoproteus and microfilariae infections. It was of moderate sensitivity (57%) and high specificity (> 90%) for Lankesterella infections, but of low sensitivity (20%) and high specificity (> 90%) for Leucocytozoon infections. Trypanosoma and Plasmodium parasites were detected only by BCM and ME, respectively. According to Cohen's kappa index, the agreement between two diagnostic tools was substantial for Haemoproteus (0.80), moderate for Lankesterella (0.46) and fair for microfilariae and Leucocytozoon (0.28) infections. Conclusions: BCM is sensitive and recommended as a quick and reliable tool to detect Haemoproteus, Trypanosoma and microfilariae parasites during fieldwork. However, it is not suitable for detection of species of Leucocytozoon and Plasmodium. BCM is a useful tool for diagnostics of blood parasite co-infections. Its application might be extended to studies of blood parasites in other vertebrates during field studies. [ABSTRACT FROM AUTHOR]

Background: Passerine birds are frequently infected with diverse haemosporidian parasites. While infections are traditionally considered benign in wild birds, recent studies demonstrated mortalities of passerine species due to exo-erythrocytic development of the parasites, which can damage organs in affected hosts. However, exo-erythrocytic development remains insufficiently investigated for most haemosporidian species and thus little is known about the virulence of tissue stages in wild passerine birds. The aim of the present study was to investigate natural haemosporidian infections in deceased Eurasian blackbirds (Turdus merula) and song thrushes (Turdus philomelos) and to determine parasite burden and associated histological effects. Methods: For molecular analysis, blood and tissue samples from 306 thrushes were screened for Plasmodium, Haemoproteus and Leucocytozoon parasites by nested PCR. For the detection of parasite stages in organ samples, tissue sections were subjected to chromogenic in situ hybridization (CISH) using genus- and species-specific probes targeting the rRNAs of parasites. Exo-erythrocytic parasite burden was semi-quantitatively assessed and histological lesions were evaluated in haematoxylin–eosin-stained sections. Results: By PCR, 179 of 277 Eurasian blackbirds and 15 of 29 song thrushes were positive for haemosporidians. Parasites of all three genera were detected, with Plasmodium matutinum LINN1 and Plasmodium vaughani SYAT05 showing the highest prevalence. CISH revealed significant differences in exo-erythrocytic parasite burden between lineages in Eurasian blackbirds, with P. matutinum LINN1 frequently causing high exo-erythrocytic parasite burdens in various organs that were associated with histological alterations. Song thrushes infected with P. matutinum LINN1 and birds infected with other haemosporidian lineages showed mostly low exo-erythrocytic parasite burdens. Two Eurasian blackbirds infected with Leucocytozoon sp. TUMER01 showed megalomeronts in various organs that were associated with inflammatory reactions and necroses. Conclusion: This study suggests that P. matutinum LINN1, a common lineage among native thrushes, regularly causes high exo-erythrocytic parasite burdens in Eurasian blackbirds, which may result in disease and mortalities, indicating its high pathogenic potential. The findings further illustrate that the same parasite lineage may show different levels of virulence in related bird species which should be considered when assessing the pathogenicity of haemosporidian parasite species. Finally, the study provides evidence of virulent Leucocytozoon sp. TUMER01 infections in two Eurasian blackbirds caused by megalomeront formation. [ABSTRACT FROM AUTHOR]

Metatranscriptomics is a powerful method for studying the composition and function of complex microbial communities. The application of metatranscriptomics to multispecies parasite infections is of particular interest, as research on parasite evolution and diversification has been hampered by technical challenges to genome‐scale DNA sequencing. In particular, blood parasites of vertebrates are abundant and diverse although they often occur at low infection intensities and exist as multispecies infections, rendering the isolation of genomic sequence data challenging. Here, we use birds and their diverse haemosporidian parasites to illustrate the potential for metatranscriptome sequencing to generate large quantities of genome‐wide sequence data from multiple blood parasite species simultaneously. We used RNA‐sequencing of 24 blood samples from songbirds in North America to show that metatranscriptomes can yield large proportions of haemosporidian protein‐coding gene repertoires even when infections are of low intensity (<0.1% red blood cells infected) and consist of multiple parasite taxa. By bioinformatically separating host and parasite transcripts and assigning them to the haemosporidian genus of origin, we found that transcriptomes detected ~23% more total parasite infections across all samples than were identified using microscopy and DNA barcoding. For single‐species infections, we obtained data for >1,300 loci from samples with as low as 0.03% parasitaemia, with the number of loci increasing with infection intensity. In total, we provide data for 1,502 single‐copy orthologous loci from a phylogenetically diverse set of 33 haemosporidian mitochondrial lineages. The metatranscriptomic approach described here has the potential to accelerate ecological and evolutionary research on haemosporidians and other diverse parasites. See also the perspective by Loren Cassin‐Sackett [ABSTRACT FROM AUTHOR]

Corticosterone (CORT) is the main glucocorticoid hormone of amphibians, reptiles, birds and some mammals. This hormone may have evolved as an adaptive metabolic mechanism, in part because increased concentrations of CORT are essential for individuals to manage energy resources and thus cope with negative perturbations such as predation and storms. The benefits of CORT are offset by costs, because elevated levels can suppress inflammatory responses of individuals, making them more susceptible to parasites and pathogens. In this study, we investigated the relationships between feather CORT levels, infection status and diversity of haemosporidian parasites in the Blue‐crowned Manakin Lepidothrix coronata, considering possible effects related to the sex and age of individuals. We predicted higher levels of feather CORT in infected individuals. We observed that feather CORT levels were similar among individuals of different sexes and ages. Although haemosporidian infection status did not vary among sexes, occurrence probability was higher among younger individuals, which may indicate that the less developed immune system of these individuals makes them more susceptible to avian malaria. Contrary to expectations, we found that feather CORT levels were not associated with the infection status and diversity of haemosporidian parasites. That haemosporidian occurrence probability does not increase with elevated feather CORT levels suggests that individuals are not immunosuppressed by elevated levels of this hormone, at least to the extent that feather CORT truly reflects individual differences in the level of this hormone. [ABSTRACT FROM AUTHOR]

Co‐infections with multiple parasite taxa are ubiquitous in nature and have the potential to impact the co‐evolutionary dynamics between host and parasite, though patterns of phylogenetic community structure of co‐infecting parasites and the processes that generate these patterns have rarely been studied across diverse host–parasite communities.Here, we tested for the roles of host and parasite evolutionary history as well as environmental variables as drivers of phylogenetic community structure among co‐infecting haemosporidian (malaria) parasites and their avian hosts in the North American boreal forest, a region characterized by an extraordinarily high blood parasite co‐infection rate.We used multiple methods to identify non‐random patterns of co‐infection among parasite species and determined whether these patterns were influenced more by co‐evolutionary host associations or environmental variables. We used model‐based approaches to test whether parasites that occurred together in a single host individual exhibited phylogenetic clustering or overdispersion. Lastly, we tested whether the observed phylogenetic community structure could be explained by parasites having convergently evolved similar patterns of host associations.We found that haemosporidian parasite co‐infections occurred at a high frequency in the boreal forest system and that parasite taxa co‐occurred in significantly non‐random patterns within host individuals and among host species. Parasite taxa that occurred in co‐infections tended to be phylogenetically overdispersed. We show that this pattern of phylogenetic overdispersion can be attributed largely to the effect of evolutionarily labile, convergent host associations that have resulted in the pool of parasites that have the potential to infect a given host consisting nearly exclusively of distantly related lineages.Our findings illustrate that environmental filtering of convergent traits can produce phylogenetically overdispersed communities, even at the level of co‐infecting parasites within an individual host organism. Broadly, this analysis illustrates how co‐evolutionary history can have a strong influence on the modern phylogenetic community assembly of diverse host–symbiont communities. [ABSTRACT FROM AUTHOR]

Avian haemosporidians are widespread and diverse and are classified in the genera Plasmodium, Haemoproteus, Leucocytozoon, and Fallisia. These species are known to cause haemosporidiosis and decreased fitness of their hosts. Despite the high diversity of habitats and animal species in Iran, only few studies have addressed avian haemosporidians in this geographic area. This study was performed in the south and southeast of Iran during the bird breeding seasons in 2017 and 2018, with the aim to partly fill in this gap. Blood samples of 237 passerine birds belonging to 41 species and 20 families were collected. Parasite infections were identified using a nested PCR protocol targeting a 479-base-pair fragment of the mitochondrial cytochrome b (cytb) gene of Haemoproteus, Plasmodium and Leucocytozoon species. The overall prevalence of haemosporidian parasites was 51.1%, and 55 different lineages were identified, of which 15 cytb lineages were new globally. The lineages of Haemoproteus predominated (63.6% of all detected lineages), followed by Leucocytozoon and Plasmodium. Nineteen new host records of haemosporidian cytb lineages were identified, and the majority of them were found in resident bird species, indicating local transmission. Thirteen co-infections (9.8% of infected individuals) of Haemoproteus and Leucocytozoon parasites in seven host species were observed. This study shows the presence of active local transmission of parasites to resident bird species in the southeast of Iran and contributes to the knowledge on haemosporidian parasite biodiversity in this poorly studied region of the world.

Abstract Background Avian malaria parasites (Plasmodium spp.) and other Haemosporida (Haemoproteus and Leucocytozoon spp.) form a diverse group of vector-transmitted blood parasites that are abundant in many bird families. Recent studies have suggested that corvids may be an important host for Plasmodium spp. and Leucocytozoon spp. Methods To investigate the diversity of Haemosporida of resident carrion crows (Corvus corone) and Eurasian Magpies (Pica pica) in southwest Germany, 100 liver samples of corvids were examined using a nested PCR method to amplify a 1063 bp fragment of the haemosporidian mitochondrial cytochrome b gene. The phylogenetic relationship of parasite lineages obtained from these birds was inferred. Results Haemosporidian DNA was detected in 85 carrion crows (89.5%) and in all five Eurasian Magpies. The most abundant parasite genus was Leucocytozoon with a prevalence of 85.3% (n = 95). 65.3% of the samples (n = 62) contained multiple infections. Thirteen haemosporidian lineages were isolated from the corvid samples. Female carrion crows were more likely infected with haemosporidian parasites than males. Discussion This study provides the first insight into the diversity of haemosporidian parasites of corvids in Germany. Very high prevalences were found and based on the applied diagnostic method also a high amount of multiple infections could be detected. Due to the high diversity of haemosporidian parasites found in corvids, they seem to be excellent model organisms to test species deliminations in haemosporidian parasites.

Abstract Background Relatively little is known about the prevalence of blood parasites in shorebirds, especially those breeding in the tropics. The prevalence of blood parasites of the genera Plasmodium, Haemoproteus and Leucocytozoon was assessed in blood samples from Kentish plovers and cream-coloured coursers in Cape Verde, and samples of Kittlitz’s plovers, Madagascar plovers and white-fronted plovers in Madagascar. Results Only two of these samples were positive for Plasmodium: a Kittlitz’s plover was infected by a generalist lineage of Plasmodium that has already been reported in Europe and Africa, while in a white-fronted plover direct sequencing revealed a previously un-described Plasmodium lineage. Conclusion Potential explanations for the low prevalence of blood parasites include the scarcity of vectors in habitats used by these bird species and their resistance to parasitic infections.

In addition to the presence of a suitable host and vector, the prevalence of haemosporidians is influenced by several important factors, including the environmental conditions of the habitat, which depend on broader geographic characteristics. The aim of this study is to perform a preliminarily assessment of the distribution of blood parasites in birds from the mountainous area of Zhongar Alatau NP and to find potential new sites for research on their ecology in Kazakhstan. The results of this research constitute the first report on the occurrence of blood parasites from this area. A total of 58 birds, from the order Passeriformes and one individual from the order Caprimulgiformes, were examined during the study. The overall prevalence of infections caused by haemosporidian parasites (Haemoproteus, Leucocytozoon) was 18.6%. Neither the genus Plasmodium nor the presence of trypanosomes and microfilariae was detected in the birds examined. Three birds (5.1% prevalence) were infected with parasites of the genus Haemoproteus, in all eleven positive birds the analyses showed the presence of parasites of the genus Leucocytozoon (18.6% prevalence). The presence of parasites genus Haemoproteus was detected only in birds that were also infected with Leucocytozoon parasites. More infections with parasites of the genus Leucocytozoon are predicted due to the higher altitude and ecological factors at the capture sites, which are more favourable for the development of vectors of this genus. The species Haemoproteus majoris was detected in the host Emberiza cioides and species Haemoproteus minutus in host Turdus merula. Other species of this genus in the hosts Cyanistes cyanus and Turdus atrogularis were not determined. The species Leucocytozoon fringilinarum was detected in the hosts Cyanistes cyanus and Parus major, Leucocytozoon dubreuili was detected in Turdus atrogularis and Turdus merula. In the other host species Aegithalos caudatus, Emberiza cioides and Periparus aterus, it was not possible to dermine the species of the genus Leucocytozoon., (© 2023. The Author(s).)

In Germany, knowledge of disease agents transmitted by arthropods in zoological gardens is scarce. In the framework of ecological studies, mosquitoes were therefore collected in German zoological gardens and examined for mosquito-borne pathogen DNA and RNA. In total, 3840 mosquitoes were screened for filarial nematodes and three groups of viruses (orthobunyaviruses, flaviviruses, alphaviruses) while 405 mosquitoes were tested for avian malaria parasites. In addition to the filarial nematode species Dirofilaria repens (n = 1) and Setaria tundra (n = 8), Sindbis virus (n = 1) and the haemosporidian genera Haemoproteus (n = 8), Leucocytozoon (n = 10) and Plasmodium (n = 1) were demonstrated. Identified pathogens have the potential to cause disease in zoo and wild animals, but some of them also in humans. Positive mosquitoes were collected most often in July, indicating the highest infection risk during this month. Most of the pathogens were found in mosquito specimens of the Culex pipiens complex, suggesting that its members possibly act as the most important vectors in the surveyed zoos, although the mere demonstration of pathogen DNA/RNA in a homogenised complete mosquito is not finally indicative for a vector role. Outcomes of the study are not only significant for arthropod management in zoological gardens, but also for the general understanding of the occurrence and spread of mosquito-borne disease agents. [ABSTRACT FROM AUTHOR]

Variation in susceptibility is ubiquitous in multi‐host, multi‐parasite assemblages, and can have profound implications for ecology and evolution in these systems. The extent to which susceptibility to parasites is phylogenetically conserved among hosts can be revealed by analysing diverse regional communities. We screened for haemosporidian parasites in 3983 birds representing 40 families and 523 species, spanning ~ 4500 m elevation in the tropical Andes. To quantify the influence of host phylogeny on infection status, we applied Bayesian phylogenetic multilevel models that included a suite of environmental, spatial, temporal, life history and ecological predictors. We found evidence of deeply conserved susceptibility across the avian tree; host phylogeny explained substantial variation in infection status, and results were robust to phylogenetic uncertainty. Our study suggests that susceptibility is governed, in part, by conserved, latent aspects of anti‐parasite defence. This demonstrates the importance of deep phylogeny for understanding present‐day ecological interactions. [ABSTRACT FROM AUTHOR]

The present study assessed the prevalence and morphology of Leucocytozoon podargii from wild tawny frogmouths (Podargus strigoides) in Western Australia (WA) and genetically characterised the cytochrome b gene (cyt b) of L. podargii in wild tawny frogmouths from WA and Queensland (QLD). The prevalence of L. podargii in wild tawny frogmouths from WA was 93.3% (14/15; 95% CI, 68.1–99.8%). The morphological characters of L. podargii from WA were similar to L. podargii from QLD: the gametocytes were round-oval shape, approximately 8–12 μm in diameter; the macrogametocytes were 12.4 μm in diameter; microgametocytes were 10.4 μm in diameter; and the ratio of macrogametocytes and microgametocytes was 3:2. Sequence analysis of partial cyt b gene fragments revealed that L. podargii sequences isolated from wild tawny frogmouths in WA shared the highest similarity (99.8% at nucleotide level and 100% at protein level) with L. podargii isolated from wild tawny frogmouths in QLD. The mitochondrial 18S rRNA gene of L. podargii gametocytes was quantified using droplet digital PCR (ddPCR), and the highest gametocyte load was detected in the lung. This finding corresponds to the results of the histological study. Based on the morphological and molecular studies, it was concluded that the Leucocytozoon parasite identified from wild tawny frogmouths in WA is consistent with L. podargii from wild tawny frogmouths in QLD, and the present study has genetically characterised two different L. podargii genotypes (QLD and WA) for the first time. [ABSTRACT FROM AUTHOR]

• Audubon's Warblers are heavily infected with diverse haemosporidian parasites. • Parasite turnover among sky islands is about three-fold higher than bird turnover. • Parasite turnover is predicted by aspects of the environment and host community. • Geographic distance among sky islands did not affect parasite community composition. • Across ranges of generalist hosts, parasites vary due to environmental filtering. Haemosporidian parasites of birds are ubiquitous in terrestrial ecosystems, but their coevolutionary dynamics remain poorly understood. If species turnover in parasites occurs at a finer scale than turnover in hosts, widespread hosts would encounter diverse parasites, potentially diversifying as a result. Previous studies have shown that some wide-ranging hosts encounter varied haemosporidian communities throughout their range, and vice-versa. More surveys are needed to elucidate mechanisms that underpin spatial patterns of diversity in this complex multi-host multi-parasite system. We sought to understand how and why a community of avian haemosporidian parasites varies in abundance and composition across elevational transects in eight sky islands in southwestern North America. We tested whether bird community composition, environment, or geographic distance explain haemosporidian parasite species turnover in a widespread host that harbors a diverse haemosporidian community, the Audubon's Warbler (Setophaga auduboni). We tested predictors of infection using generalized linear models, and predictors of bird and parasite community dissimilarity using generalized dissimilarity modeling. Predictors of infection differed by parasite genus: Parahaemoproteus was predicted by elevation and climate, Leucocytozoon varied idiosyncratically among mountains, and Plasmodium was unpredictable, but rare. Parasite turnover was nearly three-fold higher than bird turnover and was predicted by elevation, climate, and bird community composition, but not geographic distance. Haemosporidian communities vary strikingly at fine spatial scales (hundreds of kilometers), across which the bird community varies only subtly. The finer scale of turnover among parasites implies that their ranges may be smaller than those of their hosts. Avian host species should encounter different parasite species in different parts of their ranges, resulting in spatially varying selection on host immune systems. The fact that parasite turnover was predicted by bird turnover, even when considering environmental characteristics, implies that host species or their phylogenetic history plays a role in determining which parasite species will be present in a community. [ABSTRACT FROM AUTHOR]

A necropsy was conducted on a female grey-headed parrot (Poicephalus robustus suahelicus) that died following signs of depression, ruffled feathers, and inappetence. Microscopic examination revealed the presence of hemoprotozoa in the liver. A nested polymerase chain reaction (PCR), targeting the mitochondrial cytochrome b gene of Haemoproteus species, Plasmodium species, and Leucocytozoon species, was performed on frozen tissue samples collected at necropsy. The hemoprotozoa were identified by PCR analysis as Leucocytozoon species. Hemoprotozoa are rarely reported in African parrots, and this is the first report of a Leucocytozooon species infection in a Poicephalus robustus suahelicus. [ABSTRACT FROM AUTHOR]

Birds harbor a diverse group of haemosporidian parasites that reproduce and develop in the host blood cells, muscle tissue, and various organs, which can cause negative effects on the survival and reproduction of their avian hosts. Characterization of the diversity, distribution, host specificity, prevalence patterns, and phylogenetic relationships of these parasites is critical to the study of avian host–parasite ecology and evolution and for understanding and preventing epidemics in wild bird populations. Here, we tested whether muscle and liver samples collected as part of standard ornithological museum expeditions can be examined to study the diversity and distributions of haemosporidians in the same way as blood collected from individual birds that are typically banded and released. We used a standard molecular diagnostic screening method for mitochondrial DNA (cytochrome b) of the parasites and found that blood, muscle, and liver collected from the same host individual provide similar estimates of prevalence and diversity of haemosporidians from the genera Parahaemoproteus and Leucocytozoon. Although we found higher prevalence for the genus Plasmodium when we screened blood vs. liver and muscle samples, the estimates of the diversity of Plasmodium from different tissue types are not affected at the community level. Given these results, we conclude that for several reasons existing museum genetic resources collections are valuable data sources for the study of haemosporidians. First, ornithological museum collections around the world house tens of thousands of vouchered tissue samples collected from remote regions of the world. Second, the host specimens are vouchered and thus host identification and phenotype are permanently documented in databased archives with a diversity of associated ancillary data. Thus, not only can identifications be confirmed but also a diversity of morphological measurements and data can be measured and accessed for these host specimens in perpetuity. [ABSTRACT FROM AUTHOR]

Avian haemosporidians are vector-transmitted blood parasites that are distributed worldwide, abundant in many bird families and well studied across Europe and North America. Since avian haemosporidians have been poorly examined in the Palearctic migratory flyways of the Western Balkans, the goal of this study was to investigate which species of three haemosporidian genera, Plasmodium, Haemoproteus and Leucocytozoon, infect both resident and migratory passerines in Serbia. The prevalence, distribution and parasitaemia of avian haemosporidian infections were screened using both a nested PCR method and microscopy. Out of 202 birds sampled at seven localities, 66 were infected with haemosporidians, and the total prevalence was 32.7%. The great majority of infected birds (29 individuals) had moderate levels of parasitaemia. The most abundant haemosporidian genus was Haemoproteus at 26.1% prevalence. All infected birds were adults; none of the tested juveniles were infected. Mixed infection was only recorded in one bird. We identified 31 genetic lineages of haemosporidians. Two new cytochrome b lineages, of Plasmodium and Leucocytozoon, were identified and found in the hosts Common Chaffinch (Fringilla coelebs) and Golden Oriole (Oriolus oriolus), respectively. We identified three new host records for previously known lineages. The lineage GRW06 (Plasmodium elongatum) occurred in Common Chaffinch, while the lineages PARUS20 and PARUS25 (Leucocytozoon sp.) were recorded in Willow Tit (Poecile montanus) and Crested Tit (Lophophanes cristatus), respectively. We found statistically significant differences in the prevalence of three haemosporidian genera among residents and partial migrants. The difference in mean parasitaemia was significant only between residents and partial migrants. [ABSTRACT FROM AUTHOR]

Abstract Avian haemosporidian infections are widespread and can result in the decline of wild bird populations or in some cases contribute to extinction of species. We determined the prevalence and genetic diversity of avian haemosporidia in 93 samples from 22 landbird species from South Africa (N = 76) and West Africa (N = 17), of which six are intra-African migrants and one is a Palearctic migrant. The samples were analysed for the presence of avian haemosporidian DNA using real-time quantitative PCR (qPCR) and nested PCR assays targeting specific mitochondrial genes of these parasites. The cytochrome b (cyt b) gene was sequenced for all samples that tested positive and phylogenetic analysis was conducted in order to determine the relationship of the new sequences with previously published sequences from the MalAvi database. The overall prevalence of avian haemosporidiosis was 68.82% (95% CI: 56.4%–78.87%) and 82.80% (95% CI: 65.68%–86.11%) as determined by qPCR and nested PCR respectively. Eighteen (19.36%; 95% CI; 10.78%–29.97%) samples had mixed infections. Infection prevalence of all haemosporidian spp. were significantly higher (p < 0.05) in samples from West Africa. Forty-six mitochondrial sequences obtained from 14 avian species grouped into three distinct clusters of Haemoproteus (36), Leucocytozoon (8) and Plasmodium (2). These represent eight published and nine new cyt b lineages. The most common lineage was Haemoproteus sp. (VIMWE1) which was identified in two bird species from West Africa and seven bird species from South Africa. This study adds to our knowledge of host-parasite relationships of avian haemosporidia of Afrotropical birds. Graphical abstract Image 1 Highlights • Avian haemosporidian infections in free ranging birds are common and diverse. • The prevalence was 68.82% and 82.80% by qPCR and nested PCR assays respectively. • Eighteen (19.36%) samples had mixed infections. • Seventeen cytochrome b lineages were identified from 14 avian species. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Madagascar represents a biodiversity hotspot for avian Haemosporida. • 29 supposed haemosporidian species are described molecularly. • Plasmodium and Leucocytozoon seem to be generalists whereas Haemoproteus spp. might be considered specialists. • Single parasite species or lineages can deviate from this rule and should be examined individually. Abstract Previous studies about geographic patterns of species diversity of avian malaria parasites and others in the Order Haemosporida did not include the avian biodiversity hotspot Madagascar. Since there are few data available on avian malaria parasites on Madagascar, we conducted the first known large-scale molecular-based study to investigate their biodiversity. Samples (1067) from 55 bird species were examined by a PCR method amplifying nearly the whole haemosporidian cytochrome b gene (1063 bp). The parasite lineages found were further characterized phylogenetically and the degree of specialization was determined with a newly introduced host diversity index (Hd). Our results demonstrate that Madagascar indeed represents a biodiversity hotspot for avian malaria parasites as we detected 71 genetically distinct parasite lineages of the genera Plasmodium and Haemoproteus. Furthermore, by using a phylogenetic approach and including the sequence divergence we suspect that the detected haemosporidian lineages represent at least 29 groups i.e. proposed species. The here presented Hd values for each parasite regarding host species, genus and family strongly support previous works demonstrating the elastic host ranges of some avian parsites of the Order Haemosporida. Representatives of the avian parasite genera Plasmodium and Leucocytozoon tend to more often be generalists than those of the genus Haemoproteus. However, as demonstrated in various examples, there is a large overlap and single parasite lineages frequently deviate from this rule. [ABSTRACT FROM AUTHOR]

Haemosporidian parasites causing malaria-like diseases in birds are globally distributed and have been associated with reduced host fitness and mortality in susceptible bird species. This group of parasites has not only enabled a greater understanding of host specificity, virulence, and parasite dispersal, but has also been crucial in restructuring the evolutionary history of apicomplexans. Despite their importance, genomic resources of avian haemosporidians have proved difficult to obtain, and they have, as a result, been lagging behind the congeneric Plasmodium species infecting mammals. In this review, I discuss recent genomic advances in the field of avian malaria research, and outline outstanding questions that will become possible to investigate with the continued successful efforts to generate avian haemosporidian genomic data. Highlights Avian malaria research has experienced multiple challenges of generating genomic data, primarily due to host contamination. One Haemoproteus and two avian Plasmodium genome sequences have been assembled. One Haemoproteus and four avian Plasmodium transcriptome sequences have been assembled, with one incorporating gene expression levels. Targeted multilocus sequencing of specific genes has proved useful to generate phylogenetic haemosporidian data. The transcriptional responses of hosts to avian malaria infection have been investigated in only one study so far. [ABSTRACT FROM AUTHOR]

Avian malaria and related haemosporidians (Plasmodium, [Para]Haemoproteus and Leucocytoozoon) represent an exciting multihost, multiparasite system in ecology and evolution. Global research in this field accelerated after the publication in 2000 of PCR protocols to sequence a haemosporidian mitochondrial (mtDNA) barcode and the development in 2009 of an open‐access database to document the geographic and host ranges of parasite mtDNA haplotypes. Isolating haemosporidian nuclear DNA from bird hosts, however, has been technically challenging, slowing the transition to genomic‐scale sequencing techniques. We extend a recently developed sequence capture method to obtain hundreds of haemosporidian nuclear loci from wild bird samples, which typically have low levels of infection, or parasitemia. We tested 51 infected birds from Peru and New Mexico and evaluated locus recovery in light of variation in parasitemia, divergence from reference sequences and pooling strategies. Our method was successful for samples with parasitemia as low as ~0.02% (2 of 10,000 blood cells infected) and mtDNA divergence as high as 15.9% (one Leucocytozoonsample), and using the most cost‐effective pooling strategy tested. Phylogenetic relationships estimated with >300 nuclear loci were well resolved, providing substantial improvement over the mtDNA barcode. We provide protocols for sample preparation and sequence capture including custom probe sequences and describe our bioinformatics pipeline using atram 2.0, phyluce and custom Perl/Python scripts. This approach can be applied to thousands of avian samples that have already been found to have haemosporidian infections of at least moderate intensity, greatly improving our understanding of parasite speciation, biogeography and evolutionary dynamics. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • A total of 404 black flies from Thailand were molecularly screened for Leucocytozoon. • Leucocytozoon was detected in approximately 10% of the examined black flies. • Most of Leucocytozoon found are genetically similar to those in domestic chickens. • Black flies are potentially vectors of Leucocytozoon in domestic chicken. Abstract Information regarding vector-parasite association is necessary for fully understanding the epidemiology of vector borne diseases yet, this information is lacking in the case of Leucocytozoonosis in the Oriental region, despite a high incidence of the disease. In this study, we used a molecular approach based on mitochondrial cytochrome b (cyt b) sequence to detect the parasite, Leucocytozoon , in potential black fly (Simuliidae) vectors in Thailand. A total of 404 wild caught black flies representing six morphological species of two subgenera were examined.- Gomphostilbia (Simulium asakoae complex, S. chumpornense) and Simulium (S. chamlongi , S. nodosum , S. nigrogilvum). Forty-four black fly specimens from two species of the Gomphostilbia were positive for Leucocytozoon. Most (35) of these were found in a village where high numbers of domestic chicken were kept, consistent with the possibility that chickens are a host of Leucocytozoon species found in black flies. Sixteen haplotypes were identified among 44 cyt b sequences. Comparisons of the sequences with previous reports revealed that the 11 haplotypes obtained in this study were identical or very similar to unknown Leucocytozoon found in infected domestic chickens. Four haplotypes are genetically similar to L. schoutedeni and one haplotype is genetically very different from existing cyt b sequences in public database. Our results indicate that two black fly species of the subgenus Gomphostilbia in Thailand are possible vectors of Leucocytozoon transmitted among poultry and wild birds in the country. [ABSTRACT FROM AUTHOR]

Aim: The role of migratory birds in the spread of parasites is poorly known, in part because migratory strategies and behaviours potentially affecting transmission are not easy to study. We investigated the dynamics of infection by blood parasites through the annual cycle of a long‐distance Nearctic–Neotropical migratory songbird to examine the role of this species in dispersing parasites between continents. Location: The Americas. Taxon: Grey‐cheeked Thrush (Catharus minimus, Aves, Passeriformes, Turdidae), Birds. Methods: We used molecular and microscopy screening of haemosporidian parasites (Plasmodium, Haemoproteus, and Leucocytozoon) to examine the prevalence, distribution, and diversity of lineages through the annual cycle (breeding, migration, and wintering) of the grey‐cheeked thrush in North and Central America, Santa Marta mountains, the Andes, and the Amazon. We aimed to identify transmission areas, to examine the degree of sharing of haemosporidian lineages with resident birds in various areas and to assess the potential role of immunologically naïve juvenile individuals in parasite transmission. Results: Prevalence and lineage diversity of haemosporidians varied significantly over time, being higher during breeding and fall and spring migration, and declining during wintering. Grey‐cheeked thrush shared few parasite lineages with tropical resident birds and slightly more lineages with other migratory and resident boreal species. We detected gametocytes in blood during spring migration stopover, but these were of lineages not found in resident tropical birds, indicating relapses of parasites transmitted elsewhere. Transmission likely occurs mostly on the breeding grounds, where juveniles and adults carried lineages restricted to closely related species of thrushes and other species of boreal birds. Main conclusions: Long‐distance migratory songbirds are likely not important dispersers of blood parasites because there are ecological and evolutionary barriers to the interchange of parasites across vastly separated areas. Further work with thorough spatial and temporal sampling across other species, and considering the role of vectors, is necessary to understand the ecological and evolutionary factors explaining the distribution of parasites over broad scales. [ABSTRACT FROM AUTHOR]

Hemosporidian parasites rarely infect aquatic birds. Few studies have been conducted in South America identifying some lineages of the genera Plasmodium, Leucocytozoon, and Haemoproteus, but none has been done in the Neotropic cormorant (Phalacrocorax brasilianus). This species is widely distributed through the American continent, from Southern USA to Tierra del Fuego, using a wide range of aquatic habitats. We molecularly studied the occurrence and diversity of hemosporidian lineages infecting individuals of Neotropic cormorant across a broad latitudinal gradient in Chile (Arica to Tierra del Fuego). As expected, a very low occurrence of individuals infected by Plasmodium sp. (4/123, 3.3%) and Leucocytozoon sp. (2/123, 1.6%) was detected. We found no evidence of Haemoproteus sp. We identified one lineage of Plasmodium (ZEMAC01) and one new lineage of Leucocytozoon (PHABRA01) infecting cormorants. Individuals infected by Plasmodium sp. were birds from only one site (i.e., Chillán), whereas Leucocytozoon sp. was found in one bird from Valdivia and another one from Tierra del Fuego. Our results expand the known range of hemosporidian parasite lineages in aquatic birds providing an essential baseline data that contribute to a better understanding of the geographic range of hemosporidian parasites infecting Phalacrocoracidae in South America. [ABSTRACT FROM AUTHOR]

Accurate detection and identification are essential components for epidemiological, ecological, and evolutionary surveys of avian haemosporidian parasites. Microscopy has been used for more than 100 years to detect and identify these parasites; however, this technique requires considerable training and high-level expertise. Several PCR methods with highly sensitive and specific detection capabilities have now been developed in addition to microscopic examination. However, recent studies have shown that these molecular protocols are insufficient at detecting mixed infections of different haemosporidian parasite species and genetic lineages. In this study, we developed a simple, sensitive, and specific multiplex PCR assay for simultaneous detection and discrimination of parasites of the genera Plasmodium, Haemoproteus, and Leucocytozoon in single and mixed infections. Relative quantification of parasite DNA using qPCR showed that the multiplex PCR can amplify parasite DNA ranging in concentration over several orders of magnitude. The detection specificity and sensitivity of this new multiplex PCR assay were also tested in two different laboratories using previously screened natural single and mixed infections. These findings show that the multiplex PCR designed here is highly effective at identifying both single and mixed infections from all three genera of avian haemosporidian parasites. We predict that this one-step multiplex PCR assay, being convenient and inexpensive, will become a widely used method for molecular screening of avian haemosporidian parasites. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Haemosporidian infection and co-infection strongly impact both reproductive success and survival probability of great tits. • Co-infections decrease survival but increase reproductive success of birds. • The cost of co-infection on survival is higher than the cost induced by a single infection. • Co-infected birds have, however, the highest reproductive success. Abstract Theoretical studies predict that parasitic infection may impact host longevity and ultimately modify the trade-off between reproduction and survival. Indeed, a host may adjust its energy allocation in current reproduction to balance the negative effects of parasitism on its survival prospects. However, very few empirical studies tested this prediction. Avian haemosporidian parasites provide an excellent opportunity to assess the influence of parasitic infection on both host survival and reproduction. They are represented by three main genera (Plasmodium , Haemoproteus and Leucocytozoon) and are highly prevalent in many bird populations. Here we provide the first known long-term field study (12 years) to explore the effects of haemosporidian parasite infection and co-infection on fitness in two populations of great tits (Parus major), using a multistate modeling framework. We found that while co-infection decreased survival probability, both infection and co-infection increased reproductive success. This study provides evidence that co-infections can be more virulent than single infections. It also provides support for the life-history theory which predicts that reproductive effort can be adjusted to balance one's fitness when survival prospects are challenged. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Over 200 morphological character states are phylogenetically informative. • mtDNA and morphology combined retrieve a phylogeny similar to the one using the three Haemosporida genomes. • Relationships were better resolved and supported using mtDNA and morphology combined. • Subgenera Haemoproteus and Parahaemoproteus were paraphyletic. • Unique combinations of character states can taxonomically determine species of all genera except Parahaemoproteus. Abstract The traditional classification of avian Haemosporida is based mainly on morphology and life history traits. Recently, molecular hypotheses have challenged the traditional classification, leading to contradictory opinions on whether morphology is phylogenetically informative. However, the morphology has never been used to reconstruct the relationships within the group. We inferred the phylogeny of avian Haemosporida from 133 morphological characters present in blood stages. We included all species with at least one mitochondrial gene characterized (n = 93). The morphological hypothesis was compared with the one retrieved from mitochondrial DNA (mtDNA) nucleotide sequences and a hypothesis that used a combination of morphological and molecular data (i.e., total evidence). In order to recover the evolutionary history and identify phylogenetically and taxonomically informative characters, they were mapped on the total evidence phylogeny. The morphological hypothesis presented more polytomies than the other two, especially within Haemoproteus. In the molecular hypothesis, the two Haemoproteus subgenera are paraphyletic, and some relationships within Parahaemoproteus were resolved. By combining the morphological and molecular data, we were able to resolve the majority of polytomies and posterior probabilities increased. We identified a unique combination of morphological traits, clearly differentiating avian Haemosporida genera, sub-genera of Leucocytozoon and Haemoproteus , and some Plasmodium sub-genera. Plasmodium had the highest number of synapomorphies. Furthermore, 86% of the species presented a unique combination of taxonomically informative characters. A limiting factor was the mismatch of traits characterized in species descriptions, leading to a morphological matrix with a considerable amount of missing data, particularly for the stages of early young and young gametocytes (67% of all missing data). Characters lacking information for the majority of species included the colour of pigment granules, the cytoplasm appearance, and the presence and dimensions of vacuoles. According to our results, the combination of morphology and mtDNA proved to be a robust alternative to reconstruct the relationships among avian Haemosporida, obtaining a resolution and support similar to that obtained using full mitochondrial genome sequences for over 100 lineages. [ABSTRACT FROM AUTHOR]

Parasites may affect the dynamics of bird populations. Plasmodium, Leucocytozoon and Haemoproteus are well-known avian haematozoa that can trigger decreased productivity and high mortality in domesticated birds. In this study, we evaluated the prevalence of avian blood parasites (Plasmodium, Leucocytozoon and Haemoproteus) against 335 birds of 8 species in the Yazd province in central Iran. To detect blood parasites, Giemsa-stained blood smears were prepared. Of the birds, 11.64% (39/335) were infected with at least one parasite genus, particularly Haemoproteus (32.6%; 23/335). The total prevalence values for Plasmodium, Haemoproteus and Leucocytozoon were 1.7, 6.8 and 2.9%, respectively. Plasmodium had lower prevalence rates of 1.7% (6/335). Among birds, pigeons, hens and ducks have the highest prevalence of Haemoproteus, Leucocytozoon and Plasmodium parasites at 1.7%, 6.8% and 2.9%, respectively. Results from this research extend our knowledge on the incidence of avian blood parasites in domesticated birds living in central Iran. The overall low incidence of avian blood parasites in birds was found in the Yazd province, Iran.

Alien mosquito and vertebrate host species may create novel epidemiological scenarios for the transmission of pathogens naturally circulating in the invaded area. The exotic Monk parakeet (Myiopsitta monachus) has established populations in Europe and is currently considered an invasive pest. Due to their high abundance in urban areas, Monk parakeets could be involved in the transmission of pathogens, potentially affecting wildlife and livestock. To test this hypothesis, we determined the prevalence and diversity of three vector-borne parasites, namely Plasmodium, Haemoproteus and Leucocytozoon, in Monk parakeets from Barcelona. Many areas of southern Europe shelter high densities of the invasive Asian tiger mosquito Aedes albopictus, which in addition to native mosquito species could affect the transmission of mosquito-borne parasites, such as avian Plasmodium. Thus, we also sampled mosquitoes in the area to trace their blood-feeding hosts and determine the presence of Plasmodium parasites. Monk parakeets were neither infected by Plasmodium nor by Haemoproteus parasites, and only five individuals (3.13%; n = 160) were infected by Leucocytozoon. Monk parakeets were bitten by Culiseta longiareolata and represented 9.5% of Culex pipiens blood meals. The invasive Ae. albopictus showed a clear anthropophilic feeding pattern, with humans dominating its diet. Three Plasmodium lineages were detected in pools of Cx pipiens. These results suggest that Plasmodium circulating in the area cannot develop in the invasive Monk parakeet, in spite of the relatively high fraction of native mosquito vectors feeding on this species in its invaded distribution range.

Abstract Background Birds possess the most diverse assemblage of haemosporidian parasites; including three genera, Plasmodium, Haemoproteus, and Leucocytozoon. Currently there are over 200 morphologically identified avian haemosporidian species, although true species richness is unknown due to great genetic diversity and insufficient sampling in highly diverse regions. Studies aimed at surveying haemosporidian diversity involve collecting and screening samples from hundreds to thousands of individuals. Currently, screening relies on microscopy and/or single or nested standard PCR. Although effective, these methods are time and resource consuming, and in the case of microscopy require substantial expertise. Here we report a newly developed real-time PCR protocol designed to quickly and reliably detect all three genera of avian haemosporidians in a single biochemical reaction. Methods Using available DNA sequences from avian haemosporidians we designed primers R330F and R480RL, which flank a 182 base pair fragment of mitochondrial conserved rDNA. These primers were initially tested using real-time PCR on samples from Malawi, Africa, previously screened for avian haemosporidians using traditional nested PCR. Our real time protocol was further tested on 94 samples from the Cerrado biome of Brazil, previously screened using a single PCR assay for haemosporidian parasites. These samples were also amplified using modified nested PCR protocols, allowing for comparisons between the three different screening methods (single PCR, nested PCR, real-time PCR). Results The real-time PCR protocol successfully identified all three genera of avian haemosporidians from both single and mixed infections previously detected from Malawi. There was no significant difference between the three different screening protocols used for the 94 samples from the Brazilian Cerrado (χ 2 = 0.3429, df = 2, P = 0.842). After proving effective, the real-time protocol was used to screen 2113 Brazilian samples, identifying 693 positive samples. Conclusions Our real-time PCR assay proved as effective as two widely used molecular screening techniques, single PCR and nested PCR. However, the real-time protocol has the distinct advantage of detecting all three genera in a single reaction, which significantly increases efficiency by greatly decreasing screening time and cost. Our real-time PCR protocol is therefore a valuable tool in the quickly expanding field of avian haemosporidian research.