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

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

Haemosporidia of grey crowned cranes in Rwanda.

Grey crowned cranes (Balearica regulorum) have been facing significant and long-term population declines in East Africa. Studies of Haemosporidian infections are essential to gain insight into pathogenic threats and help infer vector-host relationships, resolve parasite relationships, and support conservation efforts. As part of a program to reintroduce captive cranes in Rwanda back to their natural habitats, through health checks and initial microscopic examination, 120 grey crowned cranes were selected under suspicion of harboring Haemosporidian infections following initial peripheral blood smear examinations. Of these, 104 were infected with Haemoproteus and 3 were coinfected with Leucocytozoon as detected by PCR and microscopy. Sequencing allowed us to identify 2 distinct unreported lineages of Haemoproteus antigonis and one lineage of Leucocytozoon in the subspecies of Grey Crowned Cranes endemic to East Africa, B. r. gibbericeps. Molecularly, our two lineages of Haemoproteus antigonis differ by 32 base pairs and matched with about 95 percent identity to previously reported sequences of H. antigonis found in other species of cranes. No visible morphologic differences were found when compared to images of H. antigonis from previous studies. Our work demonstrates not only a need for increased testing within the family Gruidae, but also to investigate the possibility of cryptic speciation within the morphospecies Haemoproteus antigonis.

Detection and prevalence of Haemoproteus archilochus (Haemosporida, Haemoproteidae) in two species of California hummingbirds.

Haemosporidian blood parasites are transmitted to a wide range of avian hosts via blood-sucking dipteran vectors. Microscopy has revealed an impressive diversity of avian haemosporidia with more than 250 species described. Moreover, PCR and subsequent sequence analyses have suggested a much greater diversity of haemosporidia than morphological analyses alone. Given the importance of these parasites, very few studies have focused on the charismatic hummingbirds. To date, three Haemoproteus species (Haemoproteus archilochus, Haemoproteus trochili, and Haemoproteus witti) and one Leucocytozoon species (Leucocytozoon quynzae) have been described in blood samples taken from hummingbirds (Trochilidae). Unconfirmed Plasmodium lineages have also been detected in hummingbirds. Here, we report the detection of H. archilochus in two hummingbird species (Calypte anna and Archilochus alexandri) sampled in Northern California and perform a phylogenetic analysis of mitochondrial cytochrome b (cyt b) gene lineages. A total of 261 hummingbirds (157 C. anna, 104 A. alexandri) were sampled and screened for blood parasites using PCR and microscopy techniques. Combining both methods, 4 (2.55%) haemosporidian infections were detected in C. anna and 18 (17.31%) haemosporidian infections were detected in A. alexandri. Molecular analyses revealed four distinct H. archilocus cyt b lineages, which clustered as a monophyletic clade. No species of Plasmodium or Leucocytozoon were detected in this study, raising the possibility of specific vector associations with hummingbirds. These results provide resources for future studies of haemosporidian prevalence, diversity, and pathogenicity in California hummingbird populations.

Blood parasites in northern goshawk (Accipiter gentilis) with an emphasis to Leucocytozoon toddi.

Haemosporidians and trypanosomes of the northern goshawk (Accipiter gentilis) population in the Czech Republic were studied by morphological and molecular methods. Despite the wide distribution of these medium-large birds of prey, virtually nothing is known about their blood parasites. During a 5-year period, altogether 88 nestlings and 15 adults were screened for haemosporidians and trypanosomes by microscopic examination of blood smears and by nested PCR. Both methods revealed consistently higher prevalence of blood protists in adults, Leucocytozoon (80.0 % in adults vs. 13.6 % in nestlings), Haemoproteus (60.0 vs. 2.3 %), Plasmodium (6.7 vs. 0 %), and Trypanosoma (60.0 vs. 2.3 %). Altogether, five haemosporidian lineages were detected by cytochrome b sequencing. Two broadly distributed and host nonspecific lineages, Plasmodium (TURDUS1) and Leucocytozoon (BT2), were detected only sporadically, while three newly described northern goshawk host-specific Leucocytozoon lineages (ACGE01-03) represent the absolute majority of the haemosporidians identified by molecular methods. Our findings support evidences that in falconiform birds the Leucocytozoon toddi group is formed by several host-specific clusters, with Leucocytozoon buteonis in buzzards and Leucocytozoon mathisi in hawks. Between-year comparisons revealed that the infection status of adults remained predominantly unchanged and individuals stayed uninfected or possessed the same parasite lineages; however, two gains and one loss of blood parasite taxa were also recorded.

Apparent vector-mediated parent-to-offspring transmission in an avian malaria-like parasite.

Parasite transmission strategies strongly impact host-parasite co-evolution and virulence. However, studies of vector-borne parasites such as avian malaria have neglected the potential effects of host relatedness on the exchange of parasites. To test whether extended parental care in the presence of vectors increases the probability of transmission from parents to offspring, we used high-throughput sequencing to develop microsatellites for malaria-like Leucocytozoon parasites of a wild raptor population. We show that host siblings carry genetically more similar parasites than unrelated chicks both within and across years. Moreover, chicks of mothers of the same plumage morph carried more similar parasites than nestlings whose mothers were of different morphs, consistent with matrilineal transmission of morph-specific parasite strains. Ours is the first evidence of an association between host relatedness and parasite genetic similarity, consistent with vector-mediated parent-to-offspring transmission. The conditions for such 'quasi-vertical' transmission may be common and could suppress the evolution of pathogen virulence.

Genetic diversity, temporal dynamics, and host specificity in blood parasites of passerines in north China.

Avian blood parasites have been preliminarily studied in East Asia, but no data are available from long-term monitoring. The aim of this study was to evaluate the prevalence, genetic diversity, and temporal dynamics of Plasmodium, Haemoproteus, and Leucocytozoon in two passerine communities (one forest and one urban) in north China from 2008 to 2013, as well as the association between infected lineages and host specificities. Out of 633 birds from 40 species, 157 individuals (24.8 %) were infected; overall prevalence was 26.7 % and 16.8 % in two sites, respectively. The dominant avian blood parasite genus in the forest park changed yearly between Plasmodium and Haemoproteus, while the Leucocytozoon maintained a low infection level. Forty-four haplotypes were identified by sequencing a 432-bp fragment of the cytochrome b (cyt b) gene; more than 70 % were novel (six Plasmodium lineages, 16 Haemoproteus lineages, and nine Leucocytozoon lineages). Based on our data gathered over consecutive years, we found that the highly observed lineages of Haemoproteus showed higher host diversities than those of Plasmodium, and the most infected lineage EMEL01 (100 % identity with SGS1) take on the highest host diversity but low temporal diversity of the two genera, implying that this lineage infected a great diversity of species in certain years, but maintained a lower infection level or even disappeared in other years. The results suggest that genetic diversity of avian blood parasites in East Asia is high and provides scope for further research. In addition, compared with overall analysis, yearly prevalence monitoring is important in uncovering the temporal dynamic and host specificity variations over time.

Optimization and uncertainty assessment of a gas chromatography coupled to Orbitrap mass spectrometry method to determine organic contaminants in blood: A case study of an endangered seabird.

Birds are excellent bioindicators of environmental pollution, and blood provides information on contaminant exposure, although its analysis is challenging because of the low volumes that can be sampled. The objective of the present study was to optimize and validate a miniaturized and functional extraction and analytical method based on gas chromatography coupled to Orbitrap mass spectrometry (GCOrbitrap-MS) for the trace analysis of contaminants in avian blood. Studied compounds included 25 organochlorine pesticides (OCPs), 6 polychlorinated biphenyls (PCBs), 8 polybrominated diphenyl ethers (PBDEs) and 15 polycyclic aromatic hydrocarbons (PAHs). Four extraction and clean-up conditions were optimized and compared in terms of efficiency, accuracy, and uncertainty assessment. Extraction with hexane:dichloromethane and miniaturized Florisil pipette clean-up was the most adequate considering precision and accuracy, time, and costs, and was thereafter used to analyse 20 blood samples of a pelagic seabird, namely the Bermuda petrel (Pterodroma cahow). This species, endemic to the Northwest Atlantic, is among the most endangered seabirds of the region that in the '60 faced a decrease in the breeding success likely linked to a consistent exposure to dichloro-diphenyl-trichloroethane (DDT). Indeed, p,p'-DDE, the main DDT metabolite, was detected in all samples and ranged bewteen 1.13 and 6.87 ng/g wet weight. Other ubiquitous compounds were PCBs (ranging from 0.13 to 6.76 ng/g ww), hexachlorobenzene, and mirex, while PAHs were sporadically detected at low concentrations, and PBDEs were not present. Overall, the extraction method herein proposed allowed analysing very small blood volumes (∼ 100 µL), thus respecting ethical principles prioritising the application of less-invasive sampling protocols, fundamental when studying threatened avian species.

Blood parasites (Trypanosoma, Leucocytozoon, Haemoproteus) in the Eurasian sparrowhawk (Accipiter nisus): diversity, incidence and persistence of infection at the individual level.

BACKGROUND: A high prevalence of parasites may result from life-long persistence of infection or from high reinfection rates. We have studied blood parasites in a breeding population of the accipitrid raptor, Eurasian sparrowhawk (Accipiter nisus), to determine parasite diversity and turnover. METHODS: During this 7-year study, 210 adult Eurasian sparrowhawks breeding in the city of Prague were checked for parasites using several diagnostic methods. RESULTS: In both female and male raptors, parasites of the genus Leucocytozoon were the most prevalent (92% and 85%, respectively) followed in decreasing order of prevalence by those of genus Trypanosoma (74% and 68%, respectively) and genus Haemoproteus (46% and 16%, respectively). The prevalence of all parasites increased with age in both sexes, with the females at each respective age having the higher prevalence. There was a positive association between Haemoproteus and Leucocytozoon infections. Persistence at the individual level was higher than incidence for Trypanosoma and Haemoproteus. In the case of Leucocytozoon and Trypanosoma, most individuals probably become infected in their first year of life or even before dispersal from the nest. The detected parasites belonged to Trypanosoma avium sensu stricto, Leucocytozoon sp. (haplotypes ACNI1 and ACNI3) and Leucocytozoon mathisi (haplotype ACNI4) and two new lineages of the Haemoproteus elani complex (ACCNIS6 and ACCNIS7). Detailed analysis of parasite lineages in individuals that were repeatedly sampled revealed lineage turnover that would otherwise remain hidden. Phylogenetic analysis revealed that the detected Haemoproteus belongs to a phylogenetically distant group whose taxonomic position requires further analysis. CONCLUSIONS: All three genera of blood parasites persist in infected individuals, thus enabling sustainability of vector transmission cycles. Prevalence increases with age; however, there is a high turnover of Leucocytozoon lineages. No clear evidence of parasite-induced mortality was found, and most of the individuals were infected early in life, particularly in the case of Leucocytozoon.

First Report of Blood Parasites in Black-Capped Sparrow (Arremon abeillei) from Neotropical Dry Deciduous Forest.

Avian haemosporidian parasites (Order: Haemosporida) are a diverse group of microorganisms that spend part of their life cycle in the erythrocytes of avian hosts. Parasite presence has been confirmed in all Ecuadorian regions, but some ecosystems, such as the deciduous dry forest of the Coastal region, have not been evaluated for parasite presence. Data on presence or absence, and prevalence, of haemosporidian blood parasites were obtained from avian blood samples collected in two different dry deciduous forests in Guayaquil, Ecuador: Bosque Protector Prosperina and Área Nacional de Recreación Parque Lago. Mist netting was performed between December 2018 and September 2019; blood smears were prepared from blood taken from 35 individuals of 20 bird species and all samples were analyzed by optical microscope observation. Most species showed no evidence of parasite infection, but haemosporidia were found on a blood smear obtained from a Black-capped Sparrow, Arremon abeillei (Passerelleidae), confirming this species as a newly discovered host of blood parasites.

Molecular and morphological characterization of three new species of avian Onchocercidae (Nematoda) with emphasis on circulating microfilariae.

BACKGROUND: Blood parasites have been the subject of much research, with numerous reports of the presence of microfilariae in the peripheral blood (circulating microfilariae) of birds belonging to many orders. Current limitations in molecular characterization methods and species identification using morphological characters of circulating microfilariae are major obstacles to improving our understanding the biology of Filarioidea species, particularly in wildlife. The aim of this study was to partially fill these gaps, with particular emphasis on morphological features of microfilariae, which are the most readily accessible stages of these pathogens. METHODS: Peripheral blood samples of 206 birds belonging to genera Acrocephalus (five species) and Sylvia (five species) were examined using the buffy coat method to process the blood samples for the presence of microfilariae. Positive birds were dissected to collect adult nematodes. Microfilariae and adult nematodes were described, and sequences of their mitochondrial cytochrome c oxidase subunit I and nuclear 28S rDNA gene fragments were obtained and used for molecular characterization and Bayesian phylogenetic inferences. RESULTS: Overall prevalence of microfilariae was 2.9%. Microfilariae were found in the blood samples from six birds (2 Acrocephalus scirpaceus and 1 each of A. arundinaceus, Sylvia atricapilla, S. borin and S. curruca), which were dissected. All parasite species observed were new. Eufilaria acrocephalusi sp. n. and Eufilaria sylviae sp. n. were present in subcutaneous, peritracheal and periesophageal connective tissues in A. scirpaceus and S. borin, respectively. Splendidofilaria bartletti sp. n. was found in finger joins of S. atricapilla. Illustrations of microfilariae and adult nematodes are shown, and morphological and phylogenetic analyses identified the DNA barcode haplotypes that are associated with these species. Phylogenetic analysis places the parasites of different genera in different closely related clades. CONCLUSIONS: Adult nematode morphological characters, which have been traditionally used in the taxonomy of Filarioidea species, have a phylogenetic value. Importantly, in our study parasites of different genera were readily distinguishable based on the morphology of their microfilariae. The link between molecular and morphology data requires more study in Filarioidea species research, particularly because this approach provides new knowledge on species identity using only readily accessible blood stages (microfilariae), thereby avoiding host dissection and thus minimizing harm to wildlife during research.

Assessing the seasonal and intrinsic variability of neurotoxic and cyto-genotoxic biomarkers in blood of free-living Eleonoras' falcons.

In the present study we investigated seasonal and intrinsic variability of neurotoxic and cyto-genotoxic biomarkers in blood plasma and erythrocytes of free-living Eleonoras' falcons, captured during the pre-breeding (May of 2017 and 2018) and breeding period (September of 2017) on the Antikythira Island (Greece). Specifically, blood samples of captured birds were prepared for the determination of cholinesterase (ChEs, i.e. acetylcholinesterase/AChE and butyrylcholinesterase/ BChE) activity, as well as the formation of nuclear (i.e. the formation of micronuclei into the cells/MN, binucleated cells/BN and others), and cellular/cytoplasmic (i.e. echinocytes/EC, acanthocytes/AC and notched cells/NC) abnormalities in blood plasma and erythrocytes, respectively. Our results indicated that birds sampled in late May had higher ChE and BChE activity levels, as well as higher frequency of total nuclear abnormalities. The latter were also higher in second calendar year (2cy) birds. Cellular/cytoplasmic abnormalities were less frequent in falcons having better body condition, sampled in late May, as well as in light-morph falcons. The observed ChEs activities, as well as nuclear and cellular/cytoplasmic abnormalities revealed that Eleonora's falcons are likely to be exposed to chemical agents with neurotoxic and cyto-genotoxic potential year round, while different aspects of their biology and ecology, such as their reproductive and nutritional status, could mediate their levels. Although we encourage more sampling campaigns to verify the identified seasonal and intrinsic sources of variation in biomarkers tested, the current study enriches the existing knowledge about their usefulness in the environmental monitoring and risk assessment of migratory birds, like Eleonoras' falcon.