A new long-read mitochondrial-genome protocol (PacBio HiFi) for haemosporidian parasites: a tool for population and biodiversity studies.

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.

Assessing global drivers of parasite diversity: host diversity and body mass boost avian haemosporidian diversity.

Biodiversity varies worldwide and is influenced by multiple factors, such as environmental stability and past historical events (e.g. Panama Isthmus). At the same time, organisms with unique life histories (e.g. parasites) are subject to unique selective pressures that structure their diversity patterns. Parasites represent one of the most successful life strategies, impacting, directly and indirectly, ecosystems by cascading effects on host fitness and survival. Here, I focused on a highly diverse, prevalent and cosmopolitan group of parasites (avian haemosporidians) to investigate the main drivers (e.g. host and environmental features) of regional parasite diversity on a global scale. To do so, I compiled data from 4 global datasets on (i) avian haemosporidian (malaria and malaria-like) parasites, (ii) bird species diversity, (iii) avian functional traits and (iv) climate data. Then, using generalized least square models, I evaluated the effect of host and environmental features on haemosporidian diversity. I found that haemosporidian diversity mirrors host regional diversity and that higher host body mass increases haemosporidian diversity. On the other hand, climatic conditions had no effect on haemosporidian diversity in any model. When evaluating Leucocytozoon parasites separately, I found parasite diversity was boosted by a higher proportion of migratory hosts. In conclusion, I demonstrated that haemosporidian parasite diversity is intrinsically associated with their hosts' diversity and body mass.

High prevalence of haemosporidian parasites in Eurasian jays.

Avian haemosporidians are vector-borne parasites, infecting a great variety of birds. The order Passeriformes has the highest average infection probability; nevertheless, some common species of Passeriformes have been rather poorly studied. We investigated haemosporidians in one such species, the Eurasian jay Garrulus glandarius (Corvidae), from a forest population in Hesse, Central Germany. All individuals were infected with at least one haemosporidian genus (overall prevalence: 100%). The most common infection pattern was a mixed Haemoproteus and Leucocytozoon infection, whereas no Plasmodium infection was detected. Results on lineage diversity indicate a rather pronounced host-specificity of Haemoproteus and Leucocytozoon lineages infecting birds of the family Corvidae.

Unresolved haemosporidia of the Australian skink, Egernia stokesii.

The Australian skink Egernia stokesii had been recognised as a host of two species of Plasmodium, Plasmodium mackerrasae and P. circularis; nevertheless, molecular data are available for only a single haemosporidian species of this host. Its sequences are labelled as "Plasmodium sp." or "Plasmodium mackerrasae", but morphological characteristics of this isolate are unavailable. Phylogenetic analyses of these sequences placed them into the clade of the genus Haemocystidium. In this study, blood samples of six E. stokesii were analysed by both, molecular and microscopic methods to clarify the haemosporidia of this lizard. Application of these approaches offered discordant results. Whereas sequence analysis clustered our isolates with lizard species of Haemocystidium, morphology of blood stages is more akin to Plasmodium than Haemocystidium. However, limited sampling, indistinguishable nuclei/merozoites and risk of possible hidden presence of mixed infection prevent reliable species identification of detected parasites or their description as new species of Haemocystidium.

Identification of Haemoproteus infection in an imported grey crowned crane (Balearica regulorum) in China.

Blood parasites from the order Haemosporida infect many vertebrates and cause malaria-like diseases. In this study, a haemosporidian infection was detected in a sick grey crowned crane imported into China using a combination of morphological and molecular approaches. Blood samples were collected from the jugular vein and processed for morphological identification of infective parasites using stained blood smears and microscopy. No merogony occurs in the blood cells, and sporadic pigment granules were observed. Nested-PCR assays were employed for a molecular examination, which indicated that the cytb gene of this parasite had 94.1-94.9% identity to Haemoproteus antigonis. Subsequently, its mitochondrial genome structure was determined by high-throughput sequencing using the DNBSEQ-T7 platform. The determined structure was confirmed by the Sanger sequencing using amplicons. The mitochondrial genome obtained for this parasite exhibited a low CG content (32.0%) and possessed three protein-coding genes, encoding 1068 amino acids, which constituted 53.7% of the genome. Phylogenetic analysis indicated that this parasite clustered with Haemoproteus sp. is detected in grey crowned cranes from Africa. This parasite was likely acquired during importation of this animal; thus, strict quarantine of imported ornamental animals is required to prevent the entry of new pathogens.

Avian haemosporidians of the genera Plasmodium and Haemoproteus from resident and Neotropical migrant birds in Colombia.

Avian haemosporidians of the genera Plasmodium and Haemoproteus are a group of widely distributed blood parasites that can negatively affect the fitness of their hosts. Colombia contains the greatest diversity of birds on the planet, but knowledge about the associations between haemosporidian and its avifauna is scarce and fragmented. We collected blood samples from 255 birds (203 residents and 52 neotropical migrants) belonging to 27 families and 108 species. The study was conducted in six localities in the inter-Andean valleys of the Cauca and Magdalena rivers. Parasites of the genera Plasmodium and Haemoproteus were identified in the samples by morphological and molecular analysis of a fragment of the mitochondrial gene cyt b. Among the samples, 9.3% (n = 24) were positive for Plasmodium or Haemoproteus. Co-infection with Plasmodium and Haemoproteus was found in Red-eyed Vireo. Seventeen haemosporidian lineages were identified, five of which were reported for the first time in resident birds (Common Ground Dove, Checker-throated Stipplethroat, Tropical Kingbird, Pale-breasted Thrush, and Ruddy-breasted Seedeater) and one in the Summer Tanager (neotropical migrant). The research results confirm the wide diversity of haemosporidian present in tropical lowlands and the possible role of neotropical migratory birds in dissemination on haemosporidian along their migratory routes.

First record and molecular diversity of haemosporidian parasites in Sula leucogaster (Boddaert, 1783) from the southeastern Brazilian coast.

Avian haemosporidian parasites are protozoans transmitted by vectors, belonging to the genera Plasmodium, Haemoproteus, and Leucocytozoon, capable of infecting various bird species. The prevalence of these parasites varies depending on factors such as the habitat and geographical location of the host and vector, as well as across different bird groups. Seabirds typically exhibit low infection rates due to the absence of vectors in marine environments. This study aimed to identify and characterize the molecular diversity of Haemosporida parasites in a population of Brown Boobies (Sula leucogaster) (n = 37) from the Santana Archipelago in Macaé, Rio de Janeiro state. We utilized PCR-based detection methods to confirm the presence of haemosporidian parasites and sequencing (ctyb gene) to delineate the lineages. The results uncovered a notable prevalence (48.6%) of haemosporidian parasites (Plasmodium and Haemoproteus), comprising 12 novel lineages and four previously documented lineages (establishing new host records). Our findings suggest that transmission and infection by these hemoparasites can occur both on and off the island. Overall, this study yields valuable insights into the genetic diversity of haemosporidian parasites in Brown Boobies, enriching our understanding of the ecology and epidemiology of these parasites in seabirds.

Distinct biogeographic processes and areas of endemism contributed differentially to Plasmodium and Parahaemoproteus community assembly on Marajó Island.

Amazonia is the primary source of haemosporidian diversity for South American biomes. Yet, our understanding of the contribution of each area of endemism and the biogeographical processes that generated such diversity in this group of vector transmitted parasites remains incomplete. For example, a recently formed fluvial island in the Amazon delta - Marajó Island, is composed of avian lineages from adjacent Amazonian areas of endemism, but also from open habitats, such as Cerrado. This raises the question: Is the parasite assemblage found in avian hosts on this island formed by parasite lineages from adjacent Amazonian areas of endemism or Cerrado? Here, we assessed the spatiotemporal evolution of Plasmodium and Parahaemoproteus parasites. Our biogeographic analysis showed that dispersal dominated Plasmodium diversification, whereas duplication was more frequent for the genus Parahaemoproteus. We show that the Inambari area of endemism was the primary source for Plasmodium diversity on Marajó Island, but that this island received more Parahaemoproteus lineages from Cerrado than any Amazonian area of endemism. The unique patterns of dispersal for each parasite genus coupled with their propensity to shift hosts locally may have facilitated their diversification across Amazonia, suggesting that differences in deep evolutionary history may have constrained their colonization of Marajó Island.

Host shift and natural long-distance dispersal to an oceanic island of a host-specific parasite.

Parasite dispersal and host-switching may be better understood by knowing when they occurred. We estimated when the ancestor of a parasite of great reed warblers (Acrocephalus arundinaceus) dispersed to the Seychelles and began infecting the endemic Seychelles warbler (A. sechellensis). We used mitochondrial genomes and published molecular divergence rates to estimate the date of divergence between mitochondrial haplotypes of the parasite Haemoproteus nucleocondensis (lineage GRW01) in the great reed warbler and the Seychelles warbler. We also constructed a time-calibrated phylogeny of the hosts and their relatives to determine when the ancestor of the Seychelles warbler dispersed to the Seychelles. The two GRW01 lineages diverged ca 20-451 kya, long after the ancestor of the Seychelles warbler colonized the Seychelles ca 1.76-4.36 Mya. GRW01 rarely infects other species despite apparent opportunity. Humans were likely not involved in the dispersal of this parasite because humans settled the Seychelles long after the parasite diverged from its mainland relative. Furthermore, introduced birds are unlikely hosts of GRW01. Instead, the ancestor of GRW01 may have dispersed to the Seychelles with an errant migrating great reed warbler. Our results indicate that even specialized parasites can naturally disperse long distances to become emerging infectious diseases.

Morphometric and molecular characterization of an unpigmented haemosporidian parasite in the Neotropical turnip-tailed gecko (Thecadactylus rapicauda).

Morphological traits from blood stages have been the gold standard for determining haemosporidian parasite species. However, the status of some taxa and the value of such traits in parasites from reptiles remain contentious. The scarce sampling of these species worsens the situation, and several taxa lack molecular data. A survey was performed in the Magdalena Department in Colombia, where 16 species of reptiles were captured. A peculiar haemosporidian parasite was found in the Turnip-tailed gecko Thecadactylus rapicauda. This haemosporidian does not show malarial pigment in blood stages under light microscopy; thus, it fits the Garnia genus's characters belonging to the Garniidae. However, the phylogenetic analyses using a partial sequence of cytochrome b and the mitochondrial DNA placed it within the Plasmodium clade. Our findings suggest that many putative Garnia species belong to the genus Plasmodium, like the one reported here. This study either shows that visible malarial pigment in blood stages is not a diagnostic trait of the genus Plasmodium or malarial pigment might be present in an undetectable form under a light microscope. In any case, the current taxonomy of haemosporidian parasites in reptiles requires revision. This study highlights the importance of using molecular and morphological traits to address taxonomic questions at the species and genus levels in haemosporidian parasites from reptiles.

Haemoproteus parasites and passerines: the effect of local generalists on inferences of host-parasite co-phylogeny in the British Isles.

Host­parasite associations provide a benchmark for investigating evolutionary arms races and antagonistic coevolution. However, potential ecological mechanisms underlying such associations are difficult to unravel. In particular, local adaptations of hosts and/or parasites may hamper reliable inferences of host­parasite relationships and the specialist­generalist definitions of parasite lineages, making it problematic to understand such relationships on a global scale. Phylogenetic methods were used to investigate co-phylogenetic patterns between vector-borne parasites of the genus Haemoproteus and their passeriform hosts, to infer the ecological interactions of parasites and hosts that may have driven the evolution of both groups in a local geographic domain. As several Haemoproteus lineages were only detected once, and given the occurrence of a single extreme generalist, the effect of removing individual lineages on the co-phylogeny pattern was tested. When all lineages were included, and when all singly detected lineages were removed, there was no convincing evidence for host­parasite co-phylogeny. However, when only the generalist lineage was removed, strong support for co-phylogeny was indicated, and ecological interactions could be successfully inferred. This study exemplifies the importance of identifying locally abundant lineages when sampling host­parasite systems, to provide reliable insights into the precise mechanisms underlying host­parasite interactions.

Leucocytozoon cariamae n. sp. and Haemoproteus pulcher coinfection in Cariama cristata (Aves: Cariamiformes): first mitochondrial genome analysis and morphological description of a leucocytozoid in Brazil.

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.

Avian haemosporidians of breeding birds in the Davis Mountains sky-islands of west Texas, USA.

Avian haemosporidians are protozoan parasites transmitted by insect vectors that infect birds worldwide, negatively impacting avian fitness and survival. However, the majority of haemosporidian diversity remains undescribed. Quantifying this diversity is critical to determining parasite­host relationships and host-switching potentials of parasite lineages as climate change induces both host and vector range shifts. In this study, we conducted a community survey of avian haemosporidians found in breeding birds on the Davis Mountains sky islands in west Texas, USA. We determined parasite abundance and host associations and compared our results to data from nearby regions. A total of 265 birds were screened and infections were detected in 108 birds (40.8%). Most positive infections were identified as Haemoproteus (36.2%), followed by Plasmodium (6.8%) and Leucocytozoon (0.8%). A total of 71 haemosporidian lineages were detected of which 39 were previously undescribed. We found that regional similarity influenced shared lineages, as a higher number of lineages were shared with avian communities in the sky islands of New Mexico compared to south Texas, the Texas Gulf Coast and central Mexico. We found that migratory status of avian host did not influence parasite prevalence, but that host phylogeny is likely an important driver.

Special issue: avian malaria.

Avian malaria parasites or haemosporidia are found in bird species worldwide. This special issue focuses on 3 most commonly studied genera: Haemoproteus, Plasmodium and Leucocytozoon. Seven research articles and reviews are provided to illustrate the breadth of knowledge of the diversity of avian malaria parasites in different regional habitats and across bird species, and the use of avian haemosporidian systems to examine host­parasite eco-evolutionary questions.

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.

Haemoproteus jenniae (Haemoproteidae, Haemosporida) infects gulls (Larus spp.) in South Africa, with redescription of Haemoproteus skuae.

Haemoproteus spp. are dipteran-borne protozoa that infect erythrocytes and reticulo-endothelial cells of birds. These parasites are not usually transmitted between birds belonging to different orders. The suborder Lari (order Charadriiformes) comprises ~170 avian species, the majority of which are aquatic, including gulls, terns, auklets, murres and skuas, among others. In spite of the diversity of this avian group, there is limited known diversity of haemosporidian parasites, with only 4 recorded Haemoproteus morphospecies thus far. We examined the blood smears of 21 kelp gulls (Larus dominicanus) captured at a breeding colony in South Africa, as well as Haemoproteus-positive archival blood smears of 15 kelp gulls and 1 Hartlaub's gull (Larus hartlaubii) sampled while under care at seabird rehabilitation facilities in South Africa. Haemoproteus sp. infection was detected in 19% of wild-caught kelp gulls. All parasites from the gulls were morphologically identified as Haemoproteus jenniae, a species previously recorded in Lari birds at the Galapagos Islands (Ecuador), Rocas Atoll (Brazil) and Poland. Gene sequencing uncovered a new cytochrome b lineage, LARDOM01, which was closely related to the previously reported H. jenniae lineage CREFUR01. Additionally, we evaluated a hapantotype blood smear of Haemoproteus skuae, which had been described infecting a brown skua (Catharacta antarctica) in South Africa. We provide a redescription of H. skuae and discuss the morphological characters distinguishing it from H. jenniae. Further research is necessary to improve our knowledge about the host and geographic distribution, health effects and phylogeny of H. jenniae and H. skuae.

Host phylogeny and seasonality shapes avian haemosporidian prevalence in a Brazilian biodiverse and dry forest: the Caatinga.

The relationships between host phylogenetics, functional traits and parasites in wildlife remain poorly understood in the Neotropics, especially in habitats with marked seasonal variation. Here, we examined the effect of seasonality and host functional traits on the prevalence of avian haemosporidians (Plasmodium and Haemoproteus) in the Brazilian Caatinga, a seasonally dry tropical forest. 933 birds were evaluated for haemosporidian infections. We found a high parasitism prevalence (51.2%), which was correlated with phylogenetic relatedness among avian species. Prevalence varied drastically among the 20 well-sampled species, ranging from 0 to 70%. Seasonality was the main factor associated with infections, but how this abiotic condition influenced parasite prevalence varied according to the host-parasite system. Plasmodium prevalence increased during the rainy season and, after excluding the large sample size of Columbiformes (n = 462/933), Plasmodium infection rate was maintained high in the wet season and showed a negative association with host body mass. No association was found between non-Columbiform bird prevalence and seasonality or body mass when evaluating both Plasmodium and Haemoproteus or only Haemoproteus infections. Parasite community was composed of 32 lineages including 7 new lineages. We evidenced that even dry domains can harbour a high prevalence and diversity of vector-borne parasites and pointed out seasonality as a ruling factor.

Prevalence of haemosporidia in Asian Glossy Starling with discovery of misbinding of Haemoproteus-specific primer to Plasmodium genera in Sarawak, Malaysian Borneo.

BACKGROUND: Plasmodium, Haemoproteus and Leucocytozoon are three mainly studied blood parasites known to cause malarial and pseudomalarial infections in avian worldwide. Although Sarawak is a biodiversity hotspot, molecular data on blood parasite diversity in birds are absent. The objective of the study is to determine the prevalence of blood parasite in Asian Glossy Starlings (AGS), an urban bird with high population density in Sarawak and to elucidate the phylogenetic relationship with other blood parasite. METHODS: Twenty-nine carcasses of juvenile AGS that were succumbed to death due to window collision were collected around the vicinity of Universiti Malaysia Sarawak. Nested-multiplex and nested PCR targeting the Cytochrome B gene were used to detect Plasmodium and Haemoproteus, and Leucocytozoon respectively. Two primer sets were used for Haemoproteus detection to increase detection sensitivity, with one being a genus-specific primer. RESULTS: Fourteen samples (prevalence rate: 48.28%) were found positive for avian Plasmodium. Phylogenetic analysis divided our sequences into five lineages, pFANTAIL01, pCOLL4, pACCBAD01, pALPSIS01 and pALPSIS02, with two lineages being novel. No Haemoproteus and Leucocytozoon was found in this study. However, Haemoproteus-specific primer used amplified our Plasmodium samples, making the primer non-specific to Haemoproteus only. CONCLUSION: This is the first blood parasite detection study on AGS using carcasses and blood clot as sample source in Sarawak. Due to the scarcity of longer sequences from regions with high genetic plasticity, usage of genus-specific primers should be validated with sequencing to ensure correct prevalence interpretation.

Molecular investigation of Haemoproteus and Plasmodium species of some raptors in Hatay province: new CytB lineages for raptors of Accipitriformes in Turkey.

Accipitriform raptors are significant indicators of biodiversity and environmental health. Currently, most of the studies on avian haemosporidian parasites are on passerine birds, and data on raptors is constricted, with similarities both around the world and in Turkey. This study aimed to investigate the presence of Haemoproteus and Plasmodium spp. in raptors by microscopy and nested PCR technique. The study material consisted of 47 accipitriform raptors (Buteo buteo: 14, Buteo rufinus: 7, Clanga pomarina: 8, Circaetus gallicus: 12, Milvus migrans: 6). The prevalence of haemosporidian infection was 12.8% (6/47, 1 from Buteo buteo, 4 from Clanga pomarina, 1 from Milvus migrans) microscopically and 14.9% (7/47) molecularly. One Circaetus gallicus, microscopically found to be negative, probably due to low parasitemia, was molecularly found to be positive. All PCR-positive amplicons were bidirectionally sequenced, and the identification of lineages of the isolates and phylogenetic analysis were performed using the MalAvi and GenBank databases. The study revealed H-MILANS02 lineage in Buteo buteo, H-MILANS02 and P-CIAE1 lineages in Clanga pomarina, P-GRW06 lineage in Circaetus gallicus, and P-RTSR1 lineage in Milvus migrans, respectively. While this study removes the uncertainty regarding the reporting of the H-MILANS02 lineage in Turkey, it is also the first report to reveal 3 different Plasmodium spp. CytB lineages in raptors. Moreover, the fact that the P-GRW06 lineage (Plasmodium elongatum) detected in passerine birds was detected in a raptor, Circaetus gallicus, draws attention to the need for further investigations on host-parasite interaction and gives clues about the host-shifting ability of this parasite.

Comparative performance of microscopy, nested PCR, and real-time PCR for screening avian haemosporidian parasites in Afrotropical starlings (family Sturnidae).

Prevalence studies of avian haemosporidian parasites frequently use microscopy and the nested polymerase chain reaction (PCR) protocols for detecting infections. Newer PCR protocols to detect parasites are being developed, with the distinct advantage of reducing screening cost and time, as well as increasing efficiency and sensitivity. The detection ability of microscopy and nested PCR was compared against a real-time PCR (qPCR) protocol using genomic DNA extracted from 240 bird blood samples collected from three starling species (Cape Starling, the Greater Blue-eared Starling, and the Wattled Starling; family Sturnidae) in the Kruger national park, South Africa. All three protocols successfully detected avian haemosporidian parasites with the qPCR having a considerable edge against the other two methods. Fifteen unique cytochrome b lineages were identified of which seven were new lineages. Microscopy and nested PCR recorded similar prevalence (32.92% and 35.42% respectively). The qPCR protocol used here, although more sensitive (52.92% prevalence), is not able to differentiate between parasite genera but provides the opportunity to screen a large number of samples in large-scale studies within a specific region. This study recommends the development and adoption of new molecular protocols with increased sensitivity and accuracy in prevalence studies. Nevertheless, microscopy remains essential for the morphological description of parasites and for distinguishing between abortive and successful chronic infections. The PCR-based method displays the detection of the parasitic genome but does not reveal whether parasites have or will develop into a successful infection.