In the last 30 years, the area of the study of parasitism caused by blood parasite infections on wildlife has suffered an extraordinary transformation. We will review here some of the advances produced in three specific aspects: (1) Taxonomy; this field has been profoundly transformed, moving from microscopy to molecular analyses and phylogeny. (2) Impact of infections on wildlife fitness; from an initial position in which it was understood that infections by blood parasites had little or no effect on their hosts, we have moved on to the experimental demonstration of important effects on the fitness of individuals. (3) Distribution of blood parasite infections; the distribution across latitudinal and altitudinal gradients as well as the different effects of anthropic activities on the incidence of blood parasite infections have been developed importantly during the last few years. Finally, we will look at the promising future of this discipline by pointing out some developing fields of research.

The Wildlife Malaria Network (WIMANET) is an EU-COST-funded global network of researchers and stakeholders interested in wildlife malaria and related haemosporidian parasites. The network has six working groups covering a diverse range of core topics within wildlife malaria research, focusing on genetics and genomics, species identification, vectors, haematology, communities, and communication. Up to now, the network includes 229 members from 45 countries including Europe, America, Africa, and Asia, but this number is continually growing. This review outlines the aims and goals of WIMANET, providing a summary of activities and plans for each of the six working groups for the next years. The network is open to new members, and we provide details on how both new and existing members can get involved in the network and take part in activities. WIMANET provides a global platform for collaborative and innovative research, and we encourage all members of the wildlife malaria community (and beyond) to take advantage of the opportunities the network offers.

Research on the feeding preference of mosquitoes and the malaria parasites they transmit to vertebrate hosts is crucial for understanding the host–vector–parasite relationships in this complex system. In order to contribute to the growing data on these associations, and focusing on avian malaria, we collected mosquitoes at regular intervals between April and October for 2 years (2017 and 2019) in a deciduous forest and reedbeds. Collected engorged mosquitoes were identified morphologically. Total DNA was extracted from the abdomen and head–thorax parts of the vectors and was used to identify blood meal origin as well as avian haemosporidian parasites. Vertebrate hosts were identified in 75 individuals. Forty-four of the blood meals (58.7%) had avian origin, belonging to 25 bird species, 30 (40.0%) were from mammals (13 species), and one (1.3%) from a reptile. The highest number of bird hosts were recorded in Culex pipiens. Culex modestus was found to feed on the greatest variety of blood sources—birds, mammals, and а reptile, whereas Aedes vexans, Anopheles maculipennis, and Anopheles hyrcanus were predominantly mammophilic. Avian haemosporidian parasites were isolated from 11 abdominal (11.6%) and four head–thorax (4.2%) samples. The most prevalent cytochrome b lineage in abdomens was SGS1 (Plasmodium relictum (Grassi & Feletti, 1891), n = 4), whereas CXPIP23 (Plasmodium sp.) was recorded in two of the mosquito head–thoraxes. Implementing an experimental approach focused on parasite development in the vectors will be an important next step in order to confirm the validity of the observed associations.

In wild birds, a frequent and significant aspect of co-evolution is simultaneous infections of a single avian host with multiple parasites from different haemosporidian genera, or from the same genus. To date, the majority of haemosporidian phylogenetic studies have been focused on Passerines. Other bird groups, such as nocturnal species, particularly owls, even though they play an important role in ecosystems, have been largely neglected. In our study, the use of a combination of different methodologies, including Illumina sequencing and a nested PCR protocol along with blood smear examination, provided valuable insights into detecting multiple infections. Out of 101 screened long-eared owl (Asio otus), screened in Vojvodina, Serbia, the overall prevalence was 69.3%, among which 26.73% individuals carried co-infections. With a prevalence of 41.58%, the most prevalent genus was Haemoproteus. However, no correlation was found between the presence of single or multiple infections and the body condition of long-eared owls, indicating that chronic haemosporidian infections may be harmless to the hosts during the non-breeding period. Furthermore, no significant differences in prevalence between adult and young birds were found. Additionally, long-eared owls represent a new host for 5 of 10 parasite lineages identified in this study, previously documented elsewhere. For this reason, continued research during the nesting period, when vectors are active, is crucial to better understand the full haemosporidian diversity in this top predator.

Plague, a zoonotic disease caused by Yersinia pestis, remains a major public health threat in several parts of the world, including Madagascar. Factors underlying long-term persistence and emergence of the pathogen remain poorly understood. We implemented a longitudinal survey to provide insights into plague reservoir ecology within an endemic focus. Six trapping sessions (TS) were conducted in six different localities of the Ankazobe district from 2018 to 2020 in order to monitor small mammal communities. A total of 2762 individuals composed of six species (Rattus rattus, Rattus norvegicus, Mus musculus, Setifer setosus, Suncus murinus and Tenrec ecaudatus) were caught over the six successive TS. R. rattus represented 88% of all captures, with the highest relative abundances observed during the dry season (June to August 2019). None of the micromammals tested positive for the presence of Y. pestis, neither with qPCR nor bacterial culture. However, 11 seropositive individuals (6 R. rattus, 2 M. musculus and 3 S. murinus) were retrieved following ELISA, thus leading to a global seroprevalence of 0.4%. Our study highlighted the significant influence of climatic data on the seasonal variations of R. rattus abundance and suggest that black rat control should be conducted before the dry season, that is, during high reproduction period of rats, in order to reduce the number of reproducing animals and prevent subsequent increase in abundance. As three S. murinus and two M. musculus plague seropositive were identified in the present study, their potential role in plague eco-epidemiology in Madagascar should be explored further.

The invasion of cane toads (Rhinella marina) across tropical Australia has resulted in the rapid evolution of traits that enable higher rates of dispersal, and that adapt toads to hot dry climates. In anurans, a larger heart facilitates both locomotor activity and desiccation tolerance. Heart size is also often affected, either directly or indirectly, by parasite infections. To test the effects of invasion history and parasite exposure on heart size, we studied common garden-reared toads whose parents were sourced from diverse locations, and experimentally exposed them to larvae of a nematode lungworm (Rhabdias pseudosphaerocephala). Offspring of invasion-front parents had larger hearts than did conspecifics from long-established populations. Exposure to infective lungworm larvae decreased heart mass in toads from all populations. Our study suggests that cardiovascular function, like other traits, has evolved rapidly during the toad invasion; and that lungworm parasites can modify the cardiovascular function and hence aerobic capacity of their host.

Tularemia, a zoonosis caused by Francisella tularensis, is considered endemic to the Balkan Peninsula. The main reservoirs of this disease are rodents and lagomorphs, which usually show no clinical signs and are a potential disease source for other animals and humans. The presence of F. tularensis in the tissues of rodents in Serbia was examined for the first time. The animals were collected near walking tracks and places for recreation in forest areas in Belgrade. A total of 96 mice were collected in three forest locations in the autumn of 2023. Tissue samples of lungs, liver, spleen, and kidneys were taken by autopsy, and pool samples were made for all captured animals, that is, 71 striped field mice (SFM), 13 wood mice (WM), and 12 yellow-necked mice (YNM). The bacterium F. tularensis was detected by PCR, using primers that target the tul4 gene, which codes 17-kDa lipoprotein and amplifies the 400-bp product. Thirteen samples tested positive by PCR, seven of which were confirmed by sequencing to belong to F. tularensis. F. tularensis was detected in SFM animals collected from all investigated forest areas. Based on the observed results, we can conclude that F. tularensis is present in the tissues of SFM animals captured in Belgrade's forest areas.

Parasite distributions are under the influence of host occurrence and tolerable environmental conditions. In vector-borne disease systems, including avian malaria, they are further constrained by the environmental niche of the vectors. The fact that avian haemosporidians occur in a large number of migratory and non-migratory species means the role of bird migration in the global distribution of avian haemosporidians is an open question. Because avian haemosporidians refer to a large number of genetic lineages that are well characterized in terms of host and geographic range, we introduce an ecoregion pairwise analysis that sets out to explain the similarity in lineages in pairs of ecoregions as a function of the pair's similarity in migratory and non-migratory bird communities, as well as environmental conditions. We identify important roles for each of these factors in explaining the overall geographic distribution of lineages, including strong support for the role of migratory birds in moving lineages between environmentally dissimilar ecoregions.

We investigated the phenological variation of three genera of blood parasites of lizards (Schellackia, Karyolysus, and Lankesterella) using samples of 275 adult Psammodromus algirus and 138 Acanthodactylus erythrurus collected from April to September in 2021 and 2022. Both years experienced heatwaves, and 2022 had the warmest summer on historical record for the region. We captured lizards in two close areas that differed in vegetation quality due to differential human pressure. We found that Schellackia and Karyolysus more frequently infected P. algirus, whereas Lankesterella infected A. erythrurus. We fitted generalized additive models to explain parasite intensity, including the non-linear effect of phenology sorted by sex and the linear effects of body length, body condition, host sex, heterophil–lymphocyte ratio, distance to a road, and microclimate. We found a positive effect of body length on parasite intensity across parasite genera. The intensity of Schellackia was higher in female hosts, contradicting the dogma of higher male susceptibility to infections. The intensity of Lankesterella was lower closer to the road, indicating that parasite transmission success can vary at the microgeographic scale. We found a non-linear significant increase of Karyolysus across the activity period, suggesting a relaxation of the antiparasitic control upon this genus toward the end of the activity period. For Lankesterella, we observed a phenological increase only in female lizards, suggesting sexual differences in immunocompetence. We found higher intensity of Karyolysus and Lankesterella in 2022, conforming to the idea that heat waves may impair lizards' immunocompetence.

Parasites within the host body can occupy similar ecological niches and, therefore, compete for resources. Similarly to macroecological environments, within-host habitats often provide unequal access to resources for different parasitic species. Parasites that live closer to the source of nutrients likely “intercept” resources, thus inhibiting the growth of “downstream” competitors. The eyes of vertebrates are an example of such asymmetric habitats, as lenses have priority access to nutrients over the vitreous humor. However, parasitic interactions in asymmetric environments have been rarely purposefully examined. We experimentally infected fish with lens- and vitreous-dwelling parasites (Diplostomum pseudospathaceum and Tylodelphys clavata, respectively) to compare their infection success and growth in the presence/absence of each other. We found evidence of antagonistic interactions between parasites during the infection process and their growth within hosts. Each parasite species decreased the infection success of the other to a similar extent. Importantly, in accordance with our initial hypothesis, interactions between the parasites during the growth phase were asymmetric. In the presence of D. pseudospathaceum living closer to the nutrient source (in the lens), vitreous-dwelling T. clavata grew slower than in monoinfections. In contrast, the presence of T. clavata, the “downstream” parasite, did not influence the growth of the D. pseudospathaceum. Our results highlight how within-host interactions and spatial heterogeneity affect the infection success of parasites and the size structure of infracommunities. We emphasize that such “infraecological” considerations should be considered when studying parasitic communities since within-host ecological interactions can influence infection dynamics and virulence.

Various nematodes, including onchocercids, can infect mustelids and canids. However, there are few records concerning pathogenesis in wildlife species. Diagnosis is made through microscopic observation of blood. Nevertheless, a specific onchocercid assignment is difficult to achieve. In this report, through morphological analysis of microfilariae and molecular characterization using the 18S, 12S, and COI genes, two cases of microfilarial infections were confirmed, Acanthocheilonema sp. infecting tayras (Eira barbara) and Dirofilaria sp. infecting crab-eating fox (Cerdocyon thous). To the authors' knowledge, this is the first report of Acanthocheilonema infection in E. barbara. Cross-sections of adult worms (Acanthocheilonema) were associated with necrotic foci and pyogranulomatous inflammation in the lung parenchyma and pancytopenia, and elevated creatine kinase levels were also observed. Chest x-rays revealed a mixed pulmonary pattern. Meanwhile, Dirofilaria infection in C. thous produced severe generalized suppurative, fibrinous, and hemorrhagic bronchopneumonia and glomerulonephritis, with anemia, neutrophilia, monocytosis, decreased creatinine, and elevated creatine kinase observed. Additionally, findings such as hematuria, proteinuria, glucosuria, amorphous urates, and fine granular casts were observed. Rehabilitation centers provide an opportunity to monitor emerging diseases in wildlife from a One Health perspective. Zoonotic parasites in wildlife should be addressed in an integrated and multidisciplinary manner to develop action plans that incorporate ecological, social, conservation, and public health concerns. This report highlights that Acanthocheilonema sp. and Dirofilaria sp. are potential causes of pulmonary, cutaneous, and renal disease in species like tayras and crab-eating foxes. These species should be considered in management plans for filariasis, which has veterinary and public health implications.

The ongoing invasion of the zoonotic parasite Angiostrongylus cantonensis threatens humans, other mammals, and birds. In Mallorca, neurological disorders associated with this parasite are increasingly reported in free-living Algerian hedgehogs, Atelerix algirus, raising public health concerns. Experimental infections in the related species Atelerix albiventris were conducted to investigate the role of hedgehogs in the life cycle of the parasite. Captive-born hedgehogs were orally inoculated with 200 and 2000 third-stage larvae and monitored for 50 days by behavioral observations, blood tests (hematology and biochemistry), histopathology, and qPCR analysis. Baermann's larvoscopy was used to examine feces starting from day 39 post-infection (DPI). Neuropathological symptoms were only observed in the high-dose group, with larvae detected in brain tissue at 5, 15, 23, and 44 DPI. In contrast, no clinical symptoms or larvae in tissues were found in the low-dose group. Parasite DNA was present in the organs of both groups, but neurological migration occurred exclusively in the high-dose group. Blood tests showed no specific patterns indicating infection. Hedgehogs are aberrant hosts of A. cantonensis, showing severe neuropathological symptoms at high infection levels (2000 third-stage larvae), while lower doses remain asymptomatic. The parasite cannot complete its life cycle in A. albiventris hedgehogs, which do not contribute to its ecological circulation. However, hedgehogs can serve as sentinels for the spread of A. cantonensis in newly invaded regions like Mallorca. Monitoring neurological disease in wildlife could provide valuable insights into the potential spread of this zoonotic parasite in temperate latitudes.

Visually apparent diseases are valuable for investigating and monitoring the occurrence and prevalence of pathogens in wildlife populations through passive monitoring methods like camera trapping. Rumpwear, characterized by visible clinical signs of hair breakage and damage on the lumbosacral region, affects common brushtail possums (Trichosurus vulpecula) across Australia. However, the etiology of rumpwear remains unclear, and the spatiotemporal factors are understudied. This study investigated the epidemiology of rumpwear in common brushtail possums at Adamsfield, Tasmania (Australia), and predicted rumpwear distribution across the Tasmanian landscape. We visually classified images of rumpwear clinical signs in 6908 individual possums collected from a 3-year camera trapping network. Our results revealed that: (1) adults were twice as likely to show signs of rumpwear compared to young possums; (2) rumpwear occurrence increased with the relative activity of possums at a site; and (3) prevalence of rumpwear was seasonal, being lowest in May (3.2%—late autumn) and highest in December (27.1%—early summer). Collectively, these findings suggest that the occurrence of rumpwear may be density dependent, the putative etiological agent seems to be influenced by seasonal factors or site use. Additionally, a convolution neural network (CNN) was trained to identify rumpwear automatically based on the manually (human-expert) classified camera trap images. Applying the trained classifier to 38,589 brushtail possum images from across Tasmania, the CNN predicted that rumpwear is widespread, with an overall prevalence of 18.6%. This study provides new insights into rumpwear epidemiology and identified factors for further investigating within this host–pathogen system.

Avian haemosporidian parasites are responsible for avian malaria and related blood diseases, which may cause harm to various hosts. Understanding the environmental factors that affect host‒parasite associations is critical for public health. However, most studies of this topic to date have focused on pooled data from communities, while the different traits of different host species often lead to an underestimation of these factors. In this study, we investigated the phylogeographical pattern of haemosporidian parasites in a widely distributed resident bird, the Eurasian tree sparrow (Passer montanus), as well as the impact of environmental factors, including latitude, annual temperature, and humidity, on their prevalence. We observed that the prevalence of these parasites varies across sites but is generally low. Despite that, different patterns were observed for the different parasite genera. Plasmodium lineages are more widely distributed, whereas Haemoproteus are restricted to sites at lower latitudes and with lower annual temperatures, coincident with the barrier of the Yangzi River in China. This study highlights the importance of focusing on a single host when investigating variation in host‒parasite associations.

Haemosporidian parasites exhibit a wide range of vertebrate hosts and corresponding insect vectors. Among mammals, bats host the most diverse array of haemosporidians, with seven genera identified. The genus Polychromophilus is exclusive to bats and is globally linked with hematophagous flies of the genera Basilia, Nycteribia, and Penicillidia as potential vectors. In Brazil, recent molecular studies have detected Polychromophilus in bats from the Cerrado and Atlantic Forest biomes; however, its vectors in the country remained unidentified. This study analyzed the haemosporidians infection of bat flies (24 Nycteribiidae and 43 Streblidae) collected from 13 bat species in the Legado das Águas. The bat–fly associations revealed highly specialized interactions, particularly among Basilia flies and Myotis bats. Notably, a rare interaction between Megistopoda proxima and Carollia perspicillata was also observed. Two specimens (3%) of nycteribiid flies (Basilia speiseri and Basilia lindolphoi), both collected from Myotis nigricans, tested positive for infection with Polychromophilus spp. Using cytb gene sequences, we examined the phylogenetic relationships of these Polychromophilus lineages with other global lineages. We identified two haplotypes, each clustering in distinct clades within the Polychromophilus murinus group. The presence of these parasites was further confirmed by sequencing of the clpc gene from the apicoplast genome and the nuclear asl gene. This study represents the first molecular detection of Polychromophilus spp. in a vector in Brazil, 50 years after its morphological description in the salivary glands of Basilia. These findings provide novel insights into the ecological networks in host–parasite–vector interactions in a preserved neotropical environment.

Many species of avian haemosporidian parasites have been described before molecular assessment of the infections became available. This created a discrepancy between the number of parasite species described (∼300) and the available molecular information (∼5100 lineages), with numerous morphospecies having no molecular characterization and vice versa. Lethal disease was previously reported in parrots due to the abortive development of Haemoproteus species in muscular tissues. However, the disease etiology on the parasite species levels remains insufficiently understood. This study developed molecular characterization and redescription of Haemoproteus handai Maqsood, 1943 from a blossom-headed parakeet (Psittacula roseata Biwas, 1951) sampled in Thailand (1979) and deposited at the Museum national d'Histoire naturelle in Paris, France. Blood smears and histological preparations were screened, and the cryopreserved blood was used for DNA extraction and PCR tests. The host species was confirmed, and a new cytochrome b lineage hPSIROS01 was found and attributed to H. handai. Tissue stages were present in the muscular tissues of the pectoral muscles, heart, and tongue. Parrot parasite lineages appeared in different clusters. Haemoproteus species present in the cluster with hPSIROS01 also developed tissue stages mostly in muscular tissues, suggesting that phylogenies based on the partial cytochrome b gene could be used to predict pathologies. More studies are necessary to further understand the patterns of Haemoproteus species development in parrots, particularly if the parrot haemoproteids would always develop in muscular tissues, and whether they are specific or not to parrots. We encourage researchers to deposit animal samples and their parasite samples in museum collections for future research.

Avian haemosporidians are vector-borne parasites with complex transmission dynamics influenced by host ecology and environmental factors. Both prevalence and parasitemia are key measures in host–parasite studies. While prevalence reflects the proportion of infected individuals in a population, parasitemia provides insights into the intensity of infection within hosts, offering a different but complementary perspective. In this study, we used quantitative PCR (qPCR) to investigate seasonal and interannual variation in haemosporidian infection intensity among 266 individuals from six bird species in a temperate woodland in Slovakia over 3 years (2017–2019). We focused on the four most abundant taxonomic groups: Erithacus rubecula, Sylvia atricapilla, two parid species (Parus major, Cyanistes caeruleus), and two turdid species (Turdus merula, T. philomelos). Our results revealed distinct, taxon-specific seasonal trajectories. Turdid species showed similar unimodal summer peaks, whereas parids lacked early spring infections and exhibited a steady decline from summer to autumn. E. rubecula demonstrated a gradual increase across the season, while S. atricapilla showed a bimodal pattern with peaks in late spring and early autumn. Spring intensities were highest in 2017–2018, likely due to relapse or new infections, and lowest in 2019, coinciding with warmer and wetter conditions. These findings highlight how seasonal variation in environmental conditions, such as temperature and precipitation, interacts with host life history to shape infection intensity patterns. Our study emphasizes the importance of integrating ecological context with molecular tools to better understand haemosporidian dynamics in wild bird populations.

Climate change has modified hydrometeorological patterns, influencing plague transmission risks in Inner Mongolia. Using 2013–2021 plague surveillance data from 12 regions in Inner Mongolia, we assessed drought and wet conditions’ effects on flea parasitism in two key rodents: Mongolian gerbil (Meriones unguiculatus) and Daurian ground squirrel (Spermophilus dauricus). Extreme gradient boosting (XGBoost) modeling revealed the Standardized Precipitation Evapotranspiration Index (SPEI), maximum ground surface temperature, average relative humidity, average sunshine duration, and maximum wind speed collectively explained 52.60% of generalized flea index variation (SPEI contribution: 14.13%). Distributed lag non-linear model (DLNM) analysis revealed that drought conditions cumulatively increased generalized flea indices, particularly moderate drought (SPEI = −1.8), which showed a significant lagged effect on generalized flea indices after 3 months (RR = 2.76, 95% CI: 1.78–4.27). Conversely, the cumulative effects of wet conditions were detrimental to the increase in generalized flea indices. In addition to exhibiting the generalized flea index characteristics under drought conditions, the plague vectors Nosopsyllus laeviceps and Xenopsylla conformis parasitic on M. unguiculatus showed a facilitative effect in the 3rd month following severe wet conditions, with effect sizes of relative risk (RR) = 1.97 (95% CI: 1.13–3.45) and RR = 5.96 (95% CI: 3.25–10.94), respectively. With increasing drought severity, the flea index of M. unguiculatus rose significantly after a 3-month lag, with Z-test statistics of 2.16 (SPEI_3 = −1) and 2.63 (SPEI_3 = −1.5), both p < 0.05. Under severe drought (SPEI_3 = −1.5), the cumulative RR showed a significant difference in the two rodent species (Z = 2.27, p < 0.05). Therefore, it is essential to proactively monitor drought conditions in Inner Mongolia, particularly during the 3 months following a drought, and special attention should be paid to the increased abundance of Nosopsyllus laeviceps and Xenopsylla conformis in the 3rd month following severe wet conditions.

Avian chronic hemoparasite infections occur commonly in wild birds, causing adverse effects on host fitness and breeding success. However, the potential impact of such infections on the incubation behavior has been scarcely experimentally studied. We reduced the infection of hemoparasites in wild-breeding female pied flycatchers (Ficedula hypoleuca) through medication with primaquine to test the possible effects on incubation patterns compared with non-medicated control females. As predicted, medicated females significantly reduced their parasite infection compared to control females. This had a direct significant effect on the female behavior, as medicated females were able to have longer incubation sessions, while control females reduced the time devoted to each incubation session. In addition, females from both treatment groups spent less time incubating as incubation progressed, with control females showing a greater reduction. In contrast, the average length of recess sessions did not vary across treatment groups. Moreover, incubation sessions were more frequently interrupted when clutches were smaller. However, these changes had no apparent effects on immediate fitness. To our knowledge, this is the first study showing changes in individual incubation patterns in response to parasites in a wild-bird population, adding to previous studies showing that blood parasites have detrimental effects on bird reproductive success.

Bird parasites belonging to the genus Plasmodium (Haemosporida: Plasmodiidae) are found worldwide, with over 50 described species categorized into five subgenera. The subgenus Novyella comprises 22 morphologically identified species, of which 59% are genetically associated with at least one haplotype. In the Americas, only three morphospecies have their microscopic characteristics linked to a molecular signature. In this study, we described a new species of Plasmodium (Novyella) infecting a white-shouldered fire-eye (Pyriglena leucoptera) in Brazil. Molecular analysis reveals that the new species, associated with the lineage PYLEU01, is closely genetically related to Plasmodium (Novyella) homopolare, exhibiting a genetic divergence of 4.18%. However, it differs from P. homopolare due to the presence of many mature amoeboid trophozoites and some young meronts located laterally in relation to the erythrocyte nuclei and the smaller average number of merozoites in mature erythrocytic meronts. Morphology of blood stages of new species is most similar to Plasmodium vaughani and Plasmodium rouxi, but is different from these parasites due to the presence of predominantly 4 merozoites in mature erythrocytic meronts (not characteristic of P. vaughani) and the presence of 5–6 merozoites in some mature erythrocytic meronts (not characteristic of P. rouxi). Our integrative analyses reveal that the newly described species represents a distinct Plasmodium parasite from other Novyella morphospecies.

Avian haemosporidian infections have been associated with disease outbreaks in zoos and rehabilitation centers globally. This study aimed to determine the occurrence and genetic diversity of avian haemosporidian parasites in captive birds at the National Zoological Garden in South Africa. One hundred and eighty-three blood samples from five orders and 15 species of captive flamingos, vultures, owls, ibises and parrots were analyzed for haemosporidia by nested polymerase chain reaction assays. The samples were collected as part of the zoo's studbook and archived at South African National Biodiversity Institute's Wildlife Biobank. The overall infection rate was 36.1%, and infections by Leucocytozoon spp. (33.3%) were significantly higher than Haemoproteus spp. (14.8%) (p < 0.001). Twenty-one samples (11.5%) had mixed Haemoproteus and Leucocytozoon infections. The Spotted Eagle Owl (Bubo africanus) and Barn Owl (Tyto alba) had the highest infection rates. Twenty-six sequences, similar to published sequences of Leucocytozoon spp. (lineages lCHRKLA02, lCIAE02, and lBUVIR02) and Haemoproteus spp. (lineage hTYTAL6) were obtained. Two new lineages (lBUBCAP01 and hBOSHAG02) are described in this study. This is the first molecular survey of haemosporidian parasites in captive birds of the orders Accipitriformes, Pelecaniformes, Psittaciformes, Phoenicopteriformes, and Strigiformes in South Africa. This study provides new geographical and host records of known and novel haemosporidian lineages. It highlights the need for intensive surveys of these parasites in populations of captive and free-ranging birds in South Africa, regular monitoring of infections, updated screening methods, and insect control in the zoo's animal collection to avoid disease outbreaks.