Prevalence and diversity patterns of avian blood parasites in degraded African rainforest habitats

Land use changes including deforestation, road construction and agricultural encroachments have been linked to the increased prevalence of several infectious diseases. In order to better understand how deforestation affects the prevalence of vector-borne infectious diseases in wildlife, nine paired sites were sampled (disturbed vs. undisturbed habitats) in Southern Cameroon. We studied the diversity, prevalence and distribution of avian malaria parasites ( Plasmodium spp.) and other related haemosporidians (species of Haemoproteus and Leucocytozoon ) from these sites in two widespread species of African rainforest birds, the yellow-whiskered greenbul ( Andropadus latirostris , Pycnonotidae) and the olive sunbird ( Cyanomitra olivacea , Nectariniidae). Twenty-six mitochondrial cytochrome b lineages were identified: 20 Plasmodium lineages and 6 Haemoproteus lineages. These lineages showed no geographic specificity, nor significant differences in lineage diversity between habitat types. However, we found that the prevalence of Leucocytozoon and Haemoproteus infections were significantly higher in undisturbed than in deforested habitats ( Leucocytozoon spp. 50.3% vs. 35.8%, Haemoproteus spp. 16.3% vs. 10.8%). We also found higher prevalence for all haemosporidian parasites in C. olivacea than in A. latirostris species (70.2% vs. 58.2%). Interestingly, we found one morphospecies of Plasmodium in C. olivacea , as represented by a clade of related lineages, showed increased prevalence at disturbed sites, while another showed a decrease, testifying to different patterns of transmission, even among closely related lineages of avian malaria, in relation to deforestation. Our work demonstrates that anthropogenic habitat change can affect host–parasite systems and result in opposing trends in prevalence of haemosporidian parasites in wild bird populations.     

Blood parasites of birds in Cameroon

Birds from south-central Cameroon, western Africa, were surveyed for blood parasites from August to October 1986. Of 331 birds examined, representing 65 species of 15 families and 6 orders (mostly passerines), 55 (17%) were found to be infected with 1 or more genera of hemotropic parasites. These included: Haemoproteus spp. (11% prevalence), Leucocytozoon spp. (3%), Plasmodium spp. (2%), Trypanosoma spp. (1%), and microfilariae of filariid nematodes (1%). Several new host-parasite associations were identified.     

Evidence for cryptic speciation of Leucocytozoon spp. (Haemosporida, Leucocytozoidae) in diurnal raptors

Species of Leucocytozoon (Haemosporida, Leucocytozoidae) traditionally have been described based on morphological characters of their blood stages and host cells, with limited information on their avian host specificity. Based on the current taxonomy, Leucocytozoon toddi is the sole valid species of leucocytozoids parasitizing falconiform birds. Using a nested polymerase chain reaction protocol, we determined the prevalence of Leucocytozoon infection in 5 species of diurnal raptors from California. Of 591 birds tested, 177 (29.9%) were infected with Leucocytozoon toddi. Subsequent phylogenetic analysis of the cytochrome b gene revealed that distinct haplotypes are present in hawks of these genera. Haplotypes present in Buteo spp. are not found in Accipiter spp., and there is a 10.9% sequence divergence between the 2 lineage clades. In addition, Leucocytozoon sp. from Accipiter spp. from Europe group more closely with parasites found in Accipiter spp. from California than the same California Accipiter species do with their sympatric Buteo spp. Similarly, a Leucocytozoon haplotype from a Common Buzzard (Buteo buteo) from Kazakhstan forms a monophyletic lineage with a parasite from B. jamaicensis from California. These results suggest that Leucocytozoon toddi is most likely a group of cryptic species, with 1 species infecting Buteo spp. and 1 or more species, or subspecies, infecting Accipiter spp.     

Haematozoa in Domestic Chickens and Guinea Fowl in Tanzania and Transmission of Leucocytozoon neavei and Leucocytozoon schoutedeni

SYNOPSIS. Leucocytozoon neavei and Leucocytozoon caulleryi were found in guinea fowl in Tanzania and Leucocytozoon schoutedeni in chickens. The minimum time required for sporogony of L. neavei at approximately 20 C was ∼7 days in Simulium adersi and Simulium nyasalandicum. Gametocytes of L. neavei , which appeared mature, were seen in the peripheral blood of guinea fowl chicks 14 days after experimenal inoculation with sporozoites from S. adersi or S. nyasalandicum. Sporogony of L. schoutedeni was followed in "Simulium impukane," S. adersi, Simulium vorax and S. nyasalandicum. Oocysts were ∼12 μm in diameter. Sporogony of some individuals was completed in 6–7 days at 20 C. Each oocyst produced ∼50–60 sporozoites. Gametocytes were detected in the peripheral blood of a chicken 10–14 days after experimental inoculation with sporozoites of L. schoutedeni from S. adersi and "S. impukane."     

Host specificity and incidence of Trypanosoma in some African rainforest birds: a molecular approach

Studies of host-parasite interactions in birds have contributed greatly to our understanding of the evolution and ecology of disease. Here we employ molecular techniques to determine the incidence and study the host-specificity of parasitic trypanosomes in the African avifauna. We developed a polymerase chain reaction (PCR)-based diagnostic test that amplified the small subunit ribosomal RNA gene (SSU rRNA) of Trypanosoma from avian blood samples. This nested PCR assay complements and corroborates information obtained by the traditional method of blood smear analysis. The test was used to describe the incidence of trypanosomes in 479 host individuals representing 71 rainforest bird species from Cameroon, the Ivory Coast and Equatorial Guinea. Forty-two (59%) of these potential host species harboured trypanosomes and 189 individuals (35%) were infected. To examine host and geographical specificity, we examined the morphology and sequenced a portion of the SSU rRNA gene from representative trypanosomes drawn from different hosts and collecting locations. In traditional blood smear analyses we identified two trypanosome morphospecies, T. avium and T. everetti. Our molecular and morphological results were congruent in that these two morphospecies had highly divergent SSU rRNA sequences, but the molecular assay also identified cryptic variation in T. avium, in which we found seven closely allied haplotypes. The pattern of sequence diversity within T. avium provides evidence for widespread trypanosome mixing across avian host taxa and across geographical locations. For example, T. avium lineages with identical haplotypes infected birds from different families, whereas single host species were infected by T. avium lineages with different haplotypes. Furthermore, some conspecific hosts from geographically distant sampling locations were infected with the same trypanosome lineage, but other individuals from those locations harboured different trypanosome lineages. This apparent lack of host or geographical specificity may have important consequences for the evolutionary and ecological interactions between parasitic trypanosomes and their avian hosts.     

Molecular phylogenetic analysis of circumnuclear hemoproteids (Haemosporida: Haemoproteidae) of sylviid birds, with a description of Haemoproteus parabelopolskyi sp. nov

Haemoproteus spp., with circumnuclear gametocytes and tentatively belonging to Haemoproteus belopolskyi, are widespread and prevalent in warblers belonging to the Sylviidae, with numerous mitochondrial cytochrome b (cyt b) lineages detected among them.We sampled the hemoproteids from 6 species of warblers adjacent to the Baltic Sea. Parasites were identified to species based on morphology of their gametocytes, and a segment of the parasite's cyt b gene was sequenced. Sixteen mitochondrial cyt b lineages of hemoproteids with circumnuclear gametocytes were recorded. Two clades of lineages (clade A in species of Acrocephalus and Hippolais and clade B in species of Sylvia) with sequence divergence between their lineages >5% are distinguished in the phylogenetic tree. Within the clades A and B, the genetic distance between the lineages is < or = 3.9 and < and = 2.8%, respectively. We compared the morphology of gametocytes of 3 lineages (hHIICT1, hMW1, and hSYAT2) in detail. The lineages hHIICTI and hMW1 (clade A) belong to the morphospecies H. belopolskyi. Parasites of the lineage hSYAT2 (clade B) are described as a new species Haemoproteus parabelopolskyi, which can be readily distinguished from H. belopolskyi by the significantly smaller nuclei of its macrogametocytes. Lineages closely related to H. belopolskyi and H. parabelopolskyi are identified. The sequence divergence between lineages of these 2 morphospecies ranges between 5.3 and 8.1%. It seems probable that avian Haemoproteus spp. with a genetic differentiation of > or =5% in mitochondrial cyt b gene might be morphologically differentiated at the stage of gametocytes. This study establishes the value of both PCR and morphology in identification of avian hemoproteids.     

Haemosporidian blood parasites in European birds of prey and owls

Avian blood parasites have been intensively studied using morphological methods with limited information on their host specificity and species taxonomic status. Now the analysis of gene sequences, especially the mitochondrial cytochrome b gene of the avian haemosporidian species of Haemoproteus, Plasmodium, and Leucocytozoon, offers a new tool to review the parasite specificity and status. By comparing morphological and genetic techniques, we observed nearly the same overall prevalence of haemosporidian parasites by microscopy (19.8%) and polymerase chain reaction (PCR) (21.8%) analyses. However, in contrast to the single valid Leucocytozoon species (L. toddi) in the Falconiformes we detected 4 clearly distinctive strains by PCR screening. In the Strigiformes, where the only valid Leucocytozoon species is L. danilewskyi, we detected 3 genetically different strains of Leucocytozoon spp. Two strains of Haemoproteus spp. were detected in the birds of prey and owls examined, whereas the strain found in the tawny owl belonged to the morphospecies Haemoproteus noctuae. Three Plasmodium spp. strains that had already been found in Passeriformes were also detected in the birds of prey and owls examined here, supporting previous findings indicating a broad and nonspecific host spectrum bridging different bird orders.     

Incidence and Seasonal Variations in Blood and Tissue Parasites of Yellow-Billed Magpies

SYNOPSIS. Blood and tissue smears from 156 yellow-billed magpies showed infection with hematozoa in 154 (99%). Leucocytozoon berestneffi was found in 149 (96%), Haemoproteus picae in 40 (26%), Plasmodium relictum , in 32 (21%), Trypanosoma sp. in 3 (2%), and microfilariae in 42 (27%). Fifty-two birds were infected with two genera of parasites, 27 with 3, and 4 with 4. Leucocytozoon, Plasmodium and microfilariae showed a higher incidence in tissue than in blood smears.     

Leucocytozoonosis and trypanosomiasis in redstarts in Finland.

Leucocytozoon spp. and Trypanosoma spp. blood parasites in the redstart (Phoenicurus phoenicurus) were studied during spring migration 1994 in southern Finland (53 individuals) and the breeding season 1992-1994 in northern Finland (69). Parasite prevalence was higher during the breeding season (48%) than during the migration period (13%), with no age or sex differences in the breeding site birds. In both periods, redstarts were infected by the same blood parasites Leucocytozoon shaartusicum (46% prevalence at the breeding site and 71% during the migration period) and Trypanosoma avium, complex (58% and 43%, respectively). One individual at the breeding site had contracted L. dubreuili and one at the stop-over site had T. everetti. Our results may support the assumption that tissue-hidden parasites relapse during the breeding season when birds may have diminished immune response related to egg production and brood rearing. Another explanation could be that the high abundance of ornithophilic vectors enhance parasite transmission during breeding season in northern Finland.     

Bird hosts, blood parasites and their vectors--associations uncovered by molecular analyses of blackfly blood meals

The level of host specificity of blood-sucking invertebrates may have both ecological and evolutionary implications for the parasites they are transmitting. We used blood meals from wild-caught blackflies for molecular identification of parasites and hosts to examine patterns of host specificity and how these may affect the transmission of avian blood parasites of the genus Leucocytozoon . We found that five different species of ornithophilic blackflies preferred different species of birds when taking their blood meals. Of the blackflies that contained avian blood meals, 62% were infected with Leucocytozoon parasites, consisting of 15 different parasite lineages. For the blackfly species, there was a significant association between the host width (measured as the genetic differentiation between the used hosts) and the genetic similarity of the parasites in their blood meals. The absence of similar parasite in blood meals from blackflies with different host preferences is interpreted as a result of the vector–host associations. The observed associations between blackfly species and host species are therefore likely to hinder parasites to be transmitted between different host-groups, resulting in ecologically driven associations between certain parasite lineages and hosts species.     

Additional observations on blood parasites of birds in Costa Rica

Birds from the Area de Conservacion Guanacaste in northwestern Costa Rica were surveyed for blood parasites in June 2001 and December 2001-January 2002. Of 354 birds examined, representing 141 species of 35 families and 15 orders, 44 (12.4%) were infected with blood parasites. Species of Haemoproteus (4.8% prevalence), Plasmodium (0.6%), Leucocytozoon (0.3%), Trypanosoma (2.0%), and microfilariae (7.6%) were recorded. Twelve species of birds in this survey were examined for blood parasites for the first time. Several new host-parasite associations were identified.     

Hematozoa of raptors from southern New Jersey and adjacent areas

Blood smears from 259 birds of 12 species, representing four families of raptors, from New Jersey, Pennsylvania, Delaware, and Virginia were examined for blood parasites. Infected birds constituted 59.1% of the total. Birds were infected with one or more of the following genera of protozoa: Leucocytozoon (43.2%); Haemoproteus (21.6%); Plasmodium (1.2%); and Trypanosoma (1.2%). Blood culture of 142 raptors of 11 species for Trypanosoma revealed a prevalence of 41.5%. Plasmodium circumflexum is reported for the first time in Accipiter striatus, and Trypanosoma sp. in Buteo jamaicensis.     

Blood parasites of some birds from Senegal

A total of 809 birds from Senegal, including 43 species and 21 families, were examined for hematozoans; 93 birds (11.5%) harbored blood parasites, with only 7 (7.5%) harboring mixed infections. Species of Haemoproteus occurred in 81.7% of the infected birds while species of Plasmodium. Trypanosoma, microfilaria and Leucocytozoon were encountered less frequently. The majority of the sample was composed of species of ploceids and estrildidis and blood parasites were most prevalent in the colonial-nesting ploceids. Prevalence of blood parasites in Senegal was low in comparison to that seen in birds from other parts of Africa.     

Diversity and phylogeny of mitochondrial cytochrome B lineages from six morphospecies of avian Haemoproteus (Haemosporida: Haemoproteidae)

Species of Haemoproteus (Haemosporida: Haemoproteidae), avian haemosporidians, have traditionally been described based on morphology of their gametocytes and on limited experimental information on their vertebrate host specificity. We investigated to what extent the morphological species are represented by monophyletic groups based on DNA sequence data using 2 different fragment lengths of the cytochrome b (cyt. b) gene. Phylogenetic reconstructions of obtained cyt. b lineages from 6 morphospecies of Haemoproteus showed that all lineages formed monophyletic clusters matching the morphospecies. Comparing our data with a recently published study showed that this is not always the case; the morphospecies H. belopolskyi consists of 2 distinct clusters of lineages that apparently have converged in morphology. However, the overall broad congruence between the molecular and morphological clustering of lineages will facilitate the integration of the knowledge obtained by traditional and molecular parasitology. Mean between morphospecies variation was 10-fold higher than the within species variation (5.5% vs. 0.54%), suggesting that Haemoproteus lineages with a genetic differentiation >5% are expected to be morphologically differentiated in most cases. When investigate the utility of 2 different fragment sizes of the cyt. b gene, the partial, 479-bp, cyt. b protocol picked up all mitochondrial (mt)DNA lineages that are found when using the full cyt. b gene, 1073 bp, suggesting that this protocol is sufficient for identification of most mtDNA lineages. All of the mtDNA lineages were associated with unique alleles when amplification was possible at a nuclear locus, strengthening the hypothesis that the designation of lineages based on mtDNA is largely genome-wide representative. We, therefore, propose the use of a cyt. b fragment of this length as a standard gene fragment for a DNA bar-coding system for avian Haemoproteus species.     

Prevalence of avian haemosporidian parasites and their host fidelity in the central Philippine islands

We examined the prevalence and host fidelity of avian haemosporidian parasites belonging to the genera Haemoproteus, Leucocytozoon and Plasmodium in the central Philippine islands by sampling 23 bird families (42 species). Using species-specific PCR assays of the mitochondrial cytochrome b gene (471base pairs, bp), we detected infections in 91 of the 215 screened individuals (42%). We also discriminated between single and multiple infections. Thirty-one infected individuals harbored a single Haemoproteus lineage (14%), 18 a single Leucocytozoon lineage (8%) and 12 a single Plasmodium lineage (6%). Of the 215 screened birds, 30 (14%) presented different types of multiple infections. Intrageneric mixed infections were generally more common (18 Haemoproteus/Haemoproteus, 3 Leucocytozoon/Leucocytozoon, and 1 Plasmodium/Plasmodium) than intergeneric mixed infections (7 Haemoproteus/Leucocytozoon and 1 Haemoproteus/Leucocytozoon/Plasmodium). We recovered 81 unique haemosporidian mitochondrial haplotypes. These clustered in three strongly supported monophyletic clades that correspond to the three haemosporidian genera. Related lineages of Haemoproteus and Leucocytozoon were more likely to derive from the same host family than predicted by chance; however, this was not the case for Plasmodium. These results indicate that switches between host families are more likely to occur in Plasmodium. We conclude that Haemoproteus has undergone a recent diversification across well-supported host-family specific clades, while Leucocytozoon shows a longer association with its host(s). This study supports previous evidence of a higher prevalence and stronger host-family specificity of Haemoproteus and Leucocytozoon compared to Plasmodium.     

High prevalence and diversity of blood parasites of passerine birds in Southern Turkmenistan

Thirty nine specimens of passerine birds belonging to 19 species and eight families were investigated by blood smear technique in four localities of Southern Turkmenistan in 3-18 August 1991. The overall prevalence of infection was 59%. Protists from the orders Haemosporida (genera Haemoproteus, Plasmodium, Leucocytozoon), Kinetoplastida (Trypanosoma), and Adeleida (Hepatozoon), as well as Microfilaria were found. Haemoproteids (the prevalence of infection is 44%), leucocytozoids (23%), malarial parasites (13%) and trypanosomes (13%) were most frequently recorded. Only low chronic infections (< 1% of infected cells for the great majority of intracellular parasites, and a few trypanosomes and Microfilaria in each blood smear) were seen. Haemoproteus belopolskyi, H. balmorali, H. dolniki, H. magnus, H. minutus, H. fringillae, H. majoris, Leucocytozoon dubreuili, and Trypanosoma avium were recorded for the first time in Turkmenistan. The former five above-mentioned species of haemoproteids are new records for the fauna of Middle Asia. Gametocytes of leucocytozoids in fusiform host cells were found for the first time in passerine birds in the Holarctic. The host is Parus bokharensis. Due to the wide distribution and the opportunity to collect a large parasitological material using harmless for hosts methods, bird haemosporidian parasites can be used as convenient models for ecological and evolutionary biology studies in South Turkmenistan. The heavily infected Orphean Warbler Sylvia hortensis is an especially convenient host for such purposes.     

Nonspecific patterns of vector, host and avian malaria parasite associations in a central African rainforest

Malaria parasites use vertebrate hosts for asexual multiplication and Culicidae mosquitoes for sexual and asexual development, yet the literature on avian malaria remains biased towards examining the asexual stages of the life cycle in birds. To fully understand parasite evolution and mechanism of malaria transmission, knowledge of all three components of the vector-host-parasite system is essential. Little is known about avian parasite-vector associations in African rainforests where numerous species of birds are infected with avian haemosporidians of the genera Plasmodium and Haemoproteus. Here we applied high resolution melt qPCR-based techniques and nested PCR to examine the occurrence and diversity of mitochondrial cytochrome b gene sequences of haemosporidian parasites in wild-caught mosquitoes sampled across 12 sites in Cameroon. In all, 3134 mosquitoes representing 27 species were screened. Mosquitoes belonging to four genera (Aedes, Coquillettidia, Culex and Mansonia) were infected with twenty-two parasite lineages (18 Plasmodium spp. and 4 Haemoproteus spp.). Presence of Plasmodium sporozoites in salivary glands of Coquillettidia aurites further established these mosquitoes as likely vectors. Occurrence of parasite lineages differed significantly among genera, as well as their probability of being infected with malaria across species and sites. Approximately one-third of these lineages were previously detected in other avian host species from the region, indicating that vertebrate host sharing is a common feature and that avian Plasmodium spp. vector breadth does not always accompany vertebrate-host breadth. This study suggests extensive invertebrate host shifts in mosquito-parasite interactions and that avian Plasmodium species are most likely not tightly coevolved with vector species.     

Avian hematozoa of some birds from Tchad.

A total of 389 birds of 32 species representing 14 families from Tchad were examined for blood parasites. Eighty-nine (22.9%) harbored infections of Haemoproteus (64%), Plasmodium (12.4%), Trypanosoma (1.1%), Atoxoplasma (=Lankesterella) (5.6%), and microfilaria (28.1%). Species of Leucocytozoon were not observed in the present study. The occurrence of the different genera differed markedly between bird families; members of the Ploceidae comprised 85% of the infected birds.     

Haemoproteus in Wild Turkeys from the Coastal Bend of South Texas*

SYNOPSIS. Blood smears from 133 Rio Grande wild turkeys Meleagridis gallopavo intermedia live-trapped in the Welder Wildlife Refuge in south Texas, were examined for blood parasites. All of the 63 immature turkeys and 63% of the 70 adult turkeys were infected with Haemoproteus meleagridis. The infections were light, half the cases having less than 5 infected cells per 10,000 red blood cells. Infection with H. meleagridis caused significant enlargement of the host RBC. Other parasites specifically searched for but not found were Leucocytozoon, Trypanosoma, Plasmodium and microfilariae.     

A new PCR assay for simultaneous studies of Leucocytozoon, Plasmodium, and Haemoproteus from avian blood

Many bird species host several lineages of apicomplexan blood parasites (Protista spp., Haemosporida spp.), some of which are shared across different host species. To understand such complex systems, it is essential to consider the fact that different lineages, species, and families of parasites can occur in the same population, as well as in the same individual bird, and that these parasites may compete or interact with each other. In this study, we present a new polymerase chain reaction (PCR) protocol that, for the first time, enables simultaneous typing of species from the 3 most common avian blood parasite genera (Haemoproteus, Plasmodium, and Leucocytozoon). By combining the high detection rate of a nested PCR with another PCR step to separate species of Plasmodium and Haemoproteus from Leucocytozoon, this procedure provides an easy, rapid, and accurate method to separate and investigate these parasites within a blood sample. We have applied this method to bird species with known infections of Leucocytozoon spp., Plasmodium spp., and Haemoproteus spp. To obtain a higher number of parasite lineages and to test the repeatability of the method, we also applied it to blood samples from bluethroats (Luscinia svecica), for which we had no prior knowledge regarding the blood parasite infections. Although only a small number of different bird species were investigated (6 passerine species), we found 22 different parasite species lineages (4 Haemoproteus, 8 Plasmodium, and 10 Leucocytozoon).