Maria Andreina Pacheco

Background: Malaria elimination in Latin America (LATAM) is challenged by heterogeneous Plasmodium vivax transmission intensity, yet regional genomic evidence from malaria molecular surveillance (MMS) remains scarce. We aimed to generate regional genomic evidence to inform malaria control. Methods: We analyzed 578 P. vivax isolates collected in seven LATAM countries (2010–2024) using the Pv AmpliSeq Peru targeted sequencing assay. We investigated genetic diversity, complexity of infection, population structure, relatedness and connectivity, and candidate drug-resistance variants diversity. Findings: Genetic diversity (He: 0.15–0.39) and polyclonality (0–33%) varied across LATAM. Parasite populations showed country-level and subnational structure, including marked differentiation among Loreto, Amazonas and Tumbes in Peru (FST: 0.23–0.62). Country-level connectivity was limited but occurred across the Peru–Ecuador and Peru–Brazil borders. Highly related pairs (≥90% IBD) predominated in Amazonas (80.2%), Venezuela (75.4%) and Tumbes (56.9%). At lower thresholds, parasite connections among Venezuela, Colombia, Ecuador and Tumbes, geographically coincided with the regional migration corridor from Venezuela to Peru. Candidate pvmdr1 variants for reduced chloroquine susceptibility were uncommon (3.8–5.4%), whereas antifolate resistance mutations were widespread (42.8–80.6%). Interpretation: The geographic structure suggests that P. vivax transmission is mainly maintained locally, while connectivity in some border areas indicates parasite movement between countries by human mobility. Control strategies should be tailored to local transmission settings and supported by coordinated cross-border surveillance and longitudinal sentinel MMS, particularly in hotspots and border areas.

Background: Avian haemosporidians are single-celled eukaryotic parasites of vertebrates that require dipteran vectors for transmission. The genera Plasmodium , Haemoproteus and Leucocytozoon currently comprise over 5000 parasite lineages based on a 478-bp section of the mitochondrial cytochrome b gene, which is the standard DNA barcode for avian haemosporidians. The mitochondrial genomes of apicomplexan parasites are highly condensed, with a length of approximately 6000 bp, containing three coding genes (cytochrome c oxidase subunit I, cytochrome c oxidase subunit III and cytochrome b ) and dispersed fragments of the small and large ribosomal RNA (rRNA) genes. Since the mitochondrial genomes are relatively conserved, they are valuable markers for studying the phylogenetic relationships between haemosporidian parasites. However, until recently, mitochondrial genomes were unavailable for parasites of the Haemoproteus nisi and Leucocytozoon toddi species groups, which are exclusive to accipitriform raptors and strongly diverged from other Haemoproteus and Leucocytozoon parasites. Methods: We screened 171 accipitriform raptors from Austria and Germany using new primers targeting the cytochrome b gene of a previously neglected L. toddi clade. We also developed a new primer assay that enables the amplification and sequencing of the complete mitochondrial genomes of haemosporidian parasites. This process involved long-range PCRs with lineage-specific primers placed within the cytochrome b gene, followed by five nested PCRs targeting conserved sequence regions. Results: Screening the accipitriform raptors revealed 10 new L. toddi group lineages. We sequenced 18 mitochondrial genomes belonging to five H. nisi group, nine L. toddi group, and two other Leucocytozoon lineages. Phylogenetic analyses based on mt genome sequences placed the L. toddi lineages within the genus Leucocytozoon , but the results did not support a monophyly of the genus Haemoproteus . Conclusions: The new nested PCR approach with lineage-specific primers used for the long-range PCRs described herein successfully enabled the sequencing of the complete mitochondrial genomes, even in samples with mixed infections. The mitochondrial genomes of the H. nisi and L. toddi group lineages are highly valuable for resolving the phylogenetic relationships of the order Haemosporida since these parasites belong to clades distinct from other Haemoproteus and Leucocytozoon parasites. Graphical

Haemosporidians (Haemosporida, Apicomplexa), which include malaria parasites, are found in nearly all terrestrial ecosystems. Avian haemosporidians have been extensively studied; however, there is limited information on parasites in owls (order Strigiformes). Here, haemosporidians infecting North American owls were characterized using an integrative methodology. Taking advantage of injured/dead owls from rehabilitation centres in the central and northeastern USA, 53 individuals of Bubo virginianus, Strix varia, Megascops asio, Bubo scandiacus, Aegolius acadicus, and Tyto furcata were screened using polymerase chain reaction for all samples and microscopy for those with available blood smears. Parasite mitochondrial genomes were obtained using a long-read sequencing method (PacBio HiFi), which efficiently detects multiple infections in a single host. The relationships between parasite lineages were estimated using phylogenetic and haplotype network methods. In total, 21 individuals from three species were positive by PCR: B. virginianus (14/17, 82.4%), M. asio (3/5, 60%), and S. varia (4/8, 50%). Two Plasmodium, three Haemoproteus, and four Leucocytozoon lineages were identified infecting these hosts, with one Haemoproteus and one Leucocytozoon being new to science. All positive individuals were infected with Haemoproteus parasites, and two B. virginianus had a mixed infection with Leucocytozoon and Haemoproteus species. The hSTVAR01 cytochrome b (cytb) lineage common in North America is linked for the first time to Haemoproteus syrnii. Haemoproteus syrnii was found in all 14 positive B. virginianus and two S. varia. Notably, all the cytb lineages from previously identified H. syrnii, based on erythrocytic stages, were not monophyletic, indicating the existence of an undescribed species. The pPADOM11 cytb lineage was recognized as an allele of Plasmodium elongatum. Long reads enabled the detection of mixed/co-infections. The link between genetic data and morphospecies was established in two cases. Several Leucocytozoon clades were observed; however, only one morphospecies, Leucocytozoo danilewskyi, has been described in owls. Thus, there is a need for a detailed analysis of blood stages to determine whether different owl Haemoproteus and Leucocytozoon parasites exhibit morphological differences or represent cryptic species. Overall, this study underscores the importance of high-quality molecular data in characterizing the biodiversity of haemosporidian parasites.

Background: Studies on haemosporidian diversity, including origin of human malaria parasites, malaria's zoonotic dynamic, and regional biodiversity patterns, have used target gene approaches. However, current methods have a trade-off between scalability and data quality. Here, a long-read Next-Generation Sequencing protocol using PacBio HiFi is presented. The data processing is supported by a pipeline that uses machine-learning for analysing the reads. Methods: A set of primers was designed to target approximately 6 kb, almost the entire length of the haemosporidian mitochondrial genome. Amplicons from different samples were multiplexed in an SMRTbell® library preparation. A pipeline (HmtG-PacBio Pipeline) to process the reads is also provided; it integrates multiple sequence alignments, a machine-learning algorithm that uses modified variational autoencoders, and a clustering method to identify the mitochondrial haplotypes/species in a sample. Although 192 specimens could be studied simultaneously, a pilot experiment with 15 specimens is presented, including in silico experiments where multiple data combinations were tested. Results: The primers amplified various haemosporidian parasite genomes and yielded high-quality mt genome sequences. This new protocol allowed the detection and characterization of mixed infections and co-infections in the samples. The machine-learning approach converged into reproducible haplotypes with a low error rate, averaging 0.2% per read (minimum of 0.03% and maximum of 0.46%). The minimum recommended coverage per haplotype is 30X based on the detected error rates. The pipeline facilitates inspecting the data, including a local blast against a file of provided mitochondrial sequences that the researcher can customize. Conclusions: This is not a diagnostic approach but a high-throughput method to study haemosporidian sequence assemblages and perform genotyping by targeting the mitochondrial genome. Accordingly, the methodology allowed for examining specimens with multiple infections and co-infections of different haemosporidian parasites. The pipeline enables data quality assessment and comparison of the haplotypes obtained to those from previous studies. Although a single locus approach, whole mitochondrial data provide high-quality information to characterize species pools of haemosporidian parasites.

The distribution of avian haemosporidians of the genus Leucocytozoon in the Neotropics remains poorly understood. Recent studies confirmed their presence in the region using molecular techniques alone, but evidence for gametocytes and data on putative competent hosts for Leucocytozoon are still lacking outside highland areas. We combined morphological and molecular data to characterize a new Leucocytozoon species infecting a non-migratory red-legged seriema (Cariama cristata), the first report of a competent host for Leucocytozoon in Brazil. Leucocytozoon cariamae n. sp. is distinguished from the Leucocytozoon fringillinarum group by its microgametocytes that are not strongly appressed to the host cell nucleus. The bird studied was coinfected with Haemoproteus pulcher, and we present a Bayesian phylogenetic analysis based on nearly complete mitochondrial genomes of these 2 parasites. Leucocytozoon cariamae n. sp. morphology is consistent with our phylogenetic analysis indicating that it does not share a recent common ancestor with the L. fringillinarum group. Haemoproteus pulcher and Haemoproteus catharti form a monophyletic group with Haemocystidium parasites of Reptilia, supporting the polyphyly of the genus Haemoproteus. We also discussed the hypothesis that H. pulcher and H. catharti may be avian Haemocystidium, highlighting the need to study non-passerine parasites to untangle the systematics of Haemosporida.

The Neotropics are highly diverse in avian species. Neotropical countries contribute a large part of the estimated diversity of haemosporidian parasites reported for the planet’s tropical zones. However, sampling is limited and biased, illustrated by only 30% of the genetic records (barcodes) from non-passerines, most of them not linked to a nominal species. This paper aimed to perform the molecular and morphological characterization of the haemosporidians that infect non-passerine birds from Colombia deposited in the biological collection named “Grupo de Estudio Relación Parásito Hospedero (GERPH)”. We analyzed 1239 samples from twelve biomes and two animal care facilities. Phylogenetic relationships using barcodes and mitochondrial genomes were estimated. In addition, the reports of haemosporidian infections in non-passerine birds from the Neotropics recorded after 1978 were summarized. We reported the presence of thirteen morphological haemosporidian species, four potential new species deposited in GERPH, a host range expansion for two Plasmodium species, and a barcode sequence for Haemoproteus caprimulgi. We confirmed the species associated with 56 molecular lineages reported in other neotropical countries at the genus level. Thus, biological collections and curated databases such as MalAvi are essential to support integrative approaches demanded in modern taxonomy.

Great-tailed Grackles (Quiscalus mexicanus) are a social, polygamous bird species whose populations have rapidly expanded their geographic range across North America over the past century. Before 1865, Great-tailed Grackles were only documented in Central America, Mexico, and southern Texas in the USA. Given the rapid northern expansion of this species, it is relevant to study its role in the dynamics of avian blood parasites. Here, 87 Great-tailed grackles in Arizona (a population in the new center of the range) were screened for haemosporidian parasites using microscopy and PCR targeting the parasite mitochondrial cytochrome b gene. Individuals were caught in the wild from January 2018 until February 2020. Haemosporidian parasite prevalence was 62.1% (54/87). A high Plasmodium prevalence was found (60.9%, 53/87), and one grackle was infected with Haemoproteus (Parahaemoproteus) sp. (lineage SIAMEX01). Twenty-one grackles were infected with P. cathemerium, sixteen with P. homopolare, four with P. relictum (strain GRW04), and eleven with three different genetic lineages of Plasmodium spp. that have not been characterized to species level (MOLATE01, PHPAT01, and ZEMAC01). Gametocytes were observed in birds infected with three different Plasmodium lineages, revealing that grackles are competent hosts for some parasite species. This study also suggests that grackles are highly susceptible and develop chronic infections consistent with parasite tolerance, making them competent to transmit some generalist haemosporidian lineages. It can be hypothesized that, as the Great-tailed Grackle expands its geographic range, it may affect local bird communities by increasing the transmission of local parasites but not introducing new species into the parasite species pool.

Great-tailed Grackles ( Quiscalus mexicanus ) are a social, polygamous bird species whose populations have rapidly expanded their geographic range across North America over the past century. Before 1865, Great-tailed Grackles were only documented in Central America, Mexico, and southern Texas in the USA. Given the rapid northern expansion of this species, it is relevant to study its role in the dynamics of avian blood parasites. Here, 87 Great-tailed grackles in Arizona (a population in the new center of the range) were screened for haemosporidian parasites using microscopy and PCR targeting the parasite mitochondrial cytochrome b gene. Individuals were caught in the wild from January 2018 until February 2020. Haemosporidian parasite prevalence was 60.9% (53/87). A high Plasmodium prevalence was found (59.8%, 52/87), and one grackle was infected with Haemoproteus ( Parahaemoproteus ) sp. (lineage SIAMEX01). Twenty-one grackles were infected with P. cathemerium , sixteen with P. homopolare , four with P. relictum (strain GRW04), and eleven with three different genetic lineages of Plasmodium spp. that have not been characterized to species level (MOLATE01, PHPAT01, and ZEMAC01). Gametocytes were observed in birds infected with three different Plasmodium lineages, revealing that grackles are competent hosts for some parasite species. This study also suggests that grackles are highly susceptible and develop chronic infections consistent with parasite tolerance, making them competent to transmit some generalist haemosporidian lineages. It can be hypothesized that, as the Great-tailed Grackle expands its geographic range, it may affect local bird communities by increasing the transmission of local parasites but not introducing new species into the parasite species pool.

The endangered California Condor (Gymnogyps californianus) is the largest New World Vulture in North America. Despite recovery program success in saving the species from extinction, condors remain compromised by lead poisoning and limited genetic diversity. The latter makes this species especially vulnerable to infectious diseases. Thus, taking advantage of the program of blood lead testing in Arizona, condor blood samples from 2008 to 2018 were screened for haemosporidian parasites using a nested polymerase chain reaction (PCR) protocol that targets the parasite mitochondrial cytochrome b gene. Plasmodium homopolare (Family Plasmodiidae, Order Haemosporida, Phylum Apicomplexa), was detected in condors captured in 2014 and 2017. This is the first report of a haemosporidian species infecting California Condors, and the first evidence of P. homopolare circulating in the Condor population from Arizona. Although no evidence of pathogenicity of P. homopolare in Condors was found, this study showed that the California Condors from Arizona are exposed to haemosporidian parasites that likely are spilling over from other local bird species. Thus, active surveillance should be an essential part of conservation efforts to mitigate the impact of infectious diseases, an increasingly recognized cause of global wildlife extinctions worldwide, particularly in avian populations considered vulnerable or endangered.