Molecular characterization and distribution of Haemoproteus minutus (Haemosporida,Haemoproteidae): A pathogenic avian parasite

Recently, the lineage hTURDUS2 of Haemoproteus minutus (Haemosporida, Haemoproteidae) was reported to cause mortality in captive parrots. This parasite lineage is widespread and prevalent in the blackbird Turdus merula throughout its entire distribution range. Species identity of other closely related lineages recently reported in dead parrots remains unclear, but will be important to determine for a better understanding of the epidemiology of haemoproteosis. Using polymerase chain reaction (PCR)-based and microscopic methods, we analyzed 265 blood samples collected from 52 species of wild birds in Eurasia (23 samples from Kamchatka Peninsula, 73 from Sakhalin Island, 150 from Ekaterinburg and 19 from Irkutsk regions of Russia). Single infections of the lineages hTURDUS2 (hosts are redwing Turdus iliacus and fieldfare Turdus pilaris), hTUPHI1 (song thrush Turdus philomelos) and hTUCHR01 (fieldfare, redwing, song thrush and brown-headed thrush Turdus chysolaus) were detected. We identified species of these haemoproteids based on morphology of their blood stages and conclude that these lineages belong to H. minutus, a widespread parasite of different species of thrushes (genus Turdus), which serve as reservoir hosts of this haemoproteid infection. Phylogenetic analysis shows that the lineages hTURDUS2, hTUCHR01 and hTUPHI1 of H. minutus are closely related to Haemoproteus pallidus (lineages hPFC1 and hCOLL2), Haemoproteus pallidulus (hSYAT03), and Haemoproteus sp. (hMEUND3); genetic distance among their mitochondrial cytochrome b (cyt b) lineages is small (< 1% or < 4 nucleotides). All these blood parasites are different in many morphological characters, but are similar due to one feature, which is the pale staining of their macrogametocytes' cytoplasm with Giemsa. Because of the recent publications about mortality caused by the lineages hTUPHI1 and hTURDUS2 of H. minutus in captive parrots in Europe, H. minutus and the closely related H. pallidus and H. pallidulus are worth more attention as these are possible agents of haemoproteosis in exotic birds. The present study provides barcodes for molecular detection of different lineages of H. minutus, and extends information about the distribution of this blood parasite.     

Novel Haemoproteus species (Haemosporida: Haemoproteidae) from the swallow-tailed gull (Lariidae), with remarks on the host range of hippoboscid-transmitted avian hemoproteids

Haemoproteus (Haemoproteus) jenniae n. sp. (Haemosporida: Haemoproteidae) is described from a Galapagos bird, the swallow-tailed gull Creagrus furcatus (Charadriiformes, Laridae), based on the morphology of its blood stages and segments of the mitochondrial cytochrome b (cyt b) gene. The most distinctive features of H. jenniae development are the circumnuclear gametocytes occupying all cytoplasmic space in infected erythrocytes and the presence of advanced, growing gametocytes in which the pellicle is closely appressed to the erythrocyte envelope but does not extend to the erythrocyte nucleus. This parasite is distinguishable from Haemoproteus larae, which produces similar gametocytes and parasitizes closely related species of Laridae. Haemoproteus jenniae can be distinguished from H. larae primarily due to (1) the predominantly amoeboid outline of young gametocytes, (2) diffuse macrogametocyte nuclei which do not possess distinguishable nucleoli, (3) the consistent size and shape of pigment granules, and (4) the absence of rod-like pigment granules from gametocytes. Additionally, fully-grown gametocytes of H. jenniae cause both the marked hypertrophy of infected erythrocytes in width and the rounding up of the host cells, which is not the case in H. larae. Phylogenetic analyses identified the DNA lineages that are associated with H. jenniae and showed that this parasite is more closely related to the hippoboscid-transmitted (Hippoboscidae) species than to the Culicoides spp.-transmitted (Ceratopogonidae) species of avian hemoproteids. Genetic divergence between morphologically well-differentiated H. jenniae and the hippoboscid-transmitted Haemoproteus iwa, the closely related parasite of frigatebirds (Fregatidae, Pelecaniformes), is only 0.6%; cyt b sequences of these parasites differ only by 1 base pair. This is the first example of such a small genetic difference in the cyt b gene between species of the subgenus Haemoproteus. In a segment of caseinolytic protease C gene (ClpC), genetic divergence is 4% between H. jenniae and H. iwa. This study corroborates the conclusion that hippoboscid-transmitted Haemoproteus parasites infect not only Columbiformes birds but also infect marine birds belonging to Pelecaniformes and Charadriiformes. We conclude that the vertebrate host range should be used cautiously in identification of subgenera of avian Haemoproteus species and that the phylogenies based on the cyt b gene provide evidence for determining the subgeneric position of avian hemoproteids.     

The transmission of Haemoproteus belopolskyi (Haemosporida: Haemoproteidae) of blackcap by Culicoides impunctatus (Diptera: Ceratopogonidae)

Haemoproteus belopolskyi of blackcap, Sylvia atricapilla, underwent sporogony in wild-caught female biting midges, Culicoides impunctatus, which were experimentally infected by feeding them on naturally infected birds. The engorged flies were held for 8-12 days to allow development of sporozoites and then aspirated and triturated in 0.85% saline. Seven uninfected nestlings of blackcap at the age of 20-21 days were inoculated into the pectoral muscle with 0.3 ml of the slurry containing approximately 45 sporozoites. Parasitemia of H. belopolskyi developed in 6 nestlings, with a prepatent period of 11-12 days. The maximum parasitemia varied between 0.9 and 16% of erythrocytes in different experimental hosts. Culicoides impunctatus is an experimental vector of H. belopolskyi. It is likely to be the important natural vector of Haemoproteus spp. of passerine birds in Europe.     

Phylogeny,Diversity, Distribution,and Host Specificity of Haemoproteus spp. (Apicomplexa: Haemosporida: Haemoproteidae) of Palaearctic Tortoises

A complex wide‐range study on the haemoproteid parasites of chelonians was carried out for the first time. Altogether, 811 samples from four tortoise species from an extensive area between western Morocco and eastern Afghanistan and between Romania and southern Syria were studied by a combination of microscopic and molecular‐genetic methods. Altogether 160 Haemoproteus‐positive samples were gathered in the area between central Anatolia and eastern Afghanistan. According to variability in the cytochrome b gene, two monophyletic evolutionary lineages were distinguished; by means of microscopic analysis it was revealed that they corresponded to two previously described species—Haemoproteus anatolicum and Haemoproteus caucasica. Their distribution areas overlap only in a narrow strip along the Zagros Mts. range in Iran. This fact suggests the involvement of two different vector species with separated distribution. Nevertheless, no vectors were confirmed. According to phylogenetic analyses, H. caucasica represented a sister group to H. anatolicum, and both of them were most closely related to H. pacayae and H. peltocephali, described from South American river turtles. Four unique haplotypes were revealed in the population of H. caucasica, compared with seven haplotypes in H. anatolicum. Furthermore, H. caucasica was detected in two tortoise species, Testudo graeca and Testudo horsfieldii, providing evidence that Haemoproteus is not strictly host‐specific to the tortoise host species.     

Effect of Haemoproteus belopolskyi (Haemosporida: Haemoproteidae) on body mass of the blackcap Sylvia atricapilla

The effect of initial Haemoproteus belopolskyi infection on the weight of its natural host, the blackcap Sylvia atricapilla, was investigated. Fourteen blackcap nestlings were taken at the age of 4-5 days and raised by hand in the laboratory. They were free of blood parasites. Seven 20- to 21-day-old nestlings were infected experimentally by inoculation in their pectoral muscle with approximately 45 sporozoites, which had developed in the experimentally infected biting midge Culicoides impunctatus. Seven nestlings were used as negative controls. Parasitemia developed in 6 inoculated nestlings, with a prepatent period of 11-12 days. No infections were detected in the controls during this study. The weight of experimentally infected and control birds was measured 2 days before parasitemia became patent and for a 45-day period after patency. Blood smears were prepared from all birds on the days when they were weighed. When compared with controls, there was a significant weight loss of experimentally infected blackcaps during 6 days after the decline of parasitemia at 10-16 days of patency, indicating a short-term influence of the infection on the birds' body mass. Clinical symptoms of the infection were not recorded. All birds from both groups survived until the end of the experiment.     

New avian Haemoproteus species (Haemosporida: Haemoproteidae) from African birds, with a critique of the use of host taxonomic information in hemoproteid classification

Haemoproteus (Parahaemoproteus) micronuclearis n. sp., Haemoproteus (Parahaemoproteus) nucleofascialis n. sp., Haemoproteus (Parahaemoproteus) paranucleophilus n. sp., and Haemoproteus (Parahaemoproteus) homobelopolskyi n. sp. (Haemosporida, Haemoproteidae) are described from African passeriform birds based on the morphology of their blood stages and segments of the mitochondrial cytochrome b gene. Red-billed quelea (Quelea quelea), red-headed malimbe (Malimbus rubricollis), and black-headed weaver (Ploceus melanocephalus) are the type vertebrate hosts of new hemoproteids. It is probable that new species have wide distribution in weavers in sub-Saharan Africa. Both H. micronuclearis and H. nucleofascialis can be readily distinguished from other avian hemoproteids by tiny, compact microgametocyte nuclei that are significantly smaller than macrogametocyte nuclei and are a rare character of hemosporidian parasites. Gametocytes of H. paranucleophilus are closely appressed to the erythrocyte nuclei and do not touch the erythrocyte envelope along their entire margin at all stages of their development, including fully grown gametocytes. A particularly distinctive feature of H. homobelopolskyi development is the presence of circumnuclear dumbbell-shaped macrogametocytes. Illustrations of blood stages of the new species are given, and morphological and phylogenetic analyses identify the DNA lineages that are associated with these parasites. Numerous recent studies show that some lineages of hemoproteids are often present in birds belonging to different families. As a result, the use of the host family as a taxonomic character should be questioned and preferably discouraged in hemoproteid taxonomy, particularly with regard to the parasites of passerine birds. Microscopic identification of avian hemoproteids requires comparison of Haemoproteus species described from birds of different families, as is an established practice with avian Plasmodium spp. Development of bar-coding techniques remains essential in taxonomic and field studies of hemosporidian parasites.     

Exo-erythrocytic development of two Haemoproteus species (Haemosporida,Haemoproteidae), with description of Haemoproteus dumbbellus,a new blood parasite of bunting birds (Emberizidae)

Avian haemosporidians are widespread parasites categorized into four families of the order Haemosporida (Apicomplexa). Species of the subgenus Parahaemoproteus (genus Haemoproteus) belong to the Haemoproteidae and are transmitted by Culicoides biting midges. Reports of death due to tissue damage during haemoproteosis in non-adapted birds have raised concerns about these pathogens, especially as their exo-erythrocytic development is known for only a few Haemoproteus spp. More research is needed to better understand the patterns of the parasites’ development in tissues and their impact on avian hosts. Yellowhammers Emberiza citrinella (Emberizidae) and common house martins Delichon urbicum (Hirundinidae) were screened for Haemoproteus parasites by microscopic examination of blood films and PCR-based testing. Individuals with single infection were selected for histological investigations. H & E-stained sections were screened for detection and characterization of the exo-erythrocytic stages, while chromogenic in situ hybridization (CISH) and phylogenetic analysis were performed to confirm the Haemoproteus origin and their phylogenetic relationships. Haemoproteus dumbbellus n. sp. was discovered in Emberiza citrinella single-infected with the lineage hEMCIR01. Meronts of H. dumbbellus n. sp. developed in various organs of five of six tested individuals, a pattern which was reported in other Haemoproteus species clustering in the same clade, suggesting this could be a phylogenetic trait. By contrast, in Delichon urbicum infected with the Haemoproteus lineage hDELURB2, which was linked to the more distantly related parasite Haemoproteus hirundinis, only megalomeronts were found in the pectoral muscles of two of six infected individuals. All exo-erythrocytic stages were confirmed to be Haemoproteus parasites by CISH using a Haemoproteus genus-specific probe. While the development of meronts seems to be typical for species of the clade containing H. dumbbellus, further investigations and data from more species are needed to explore whether a phylogenetic pattern occurs in meront or megalomeront formation.     

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.     

Ultrastructure of the ookinetes of Haemoproteus meleagridis (Haemosporina: Haemoproteidae)

Ookinetes of Haemoproteus meleagridis were structurally similar to kinetes of other apicomplexan parasites and possessed a polar ring complex (PRC) composed of an electron-lucent polar ring with 25 supporting tines. Fifty subpellicular microtubules were anchored in a circle to the inner surface of the polar ring. A bilayered electron-dense canopy was continuous with the inner layer of the pellicle and formed a caplike cover over the PRC. Embedded rings of actin-sized microfilaments completely encircled each layer of the canopy. Numerous micronemes, 2 smaller preconoidal rings, and a conoid composed of approximately 6 spirally wound, electron-dense tubules were also present. Other organelles were similar to those reported in previous studies of haemosporidian ookinetes. Mature ookinetes of H. meleagridis developed in the midguts of engorged specimens of Culicoides edeni (Diptera: Ceratopogonidae) within 24 hr after a blood meal. Most parasites were found beneath, or embedded within, a peritrophic membrane composed of fine granules and fibrils. The observation of actin-sized microfilaments within the canopy is a previously unrecognized modification of the pellicle that probably supports the anterior end of ookinetes during penetration of the peritrophic membrane.     

An annotated checklist of the valid avian species of Haemoproteus,Leucocytozoon (Apicomplexa: Haemosporida) and Hepatozoon (Apicomplexa: Haemogregarinidae)

The valid avian species of the apicomplexan blood parasite genera Haemoproteus, Hepatozoon and Leucocytozoon are arranged according to host family, assuming host familial specificity; salient points of the parasite morphology are recorded where appropriate.     

Molecular phylogeny of avian genus Syrmaticus based on the mitochondrial cytochrome B gene and control region

Mitochondrial DNA cytochrome b (cyt b) and control region (CR) nucleotide sequences were used to study the molecular phylogeny of the genus Syrmaticus. We found that the substitution rates among the three codon positions of cyt b were heterogeneous and the transition-transversion ratio was highly biased. As to CR sequences of the genus, most variable sites were in the peripheral domains. All molecular phylogenetic trees based on the two genes showed that: 1) the Syrmaticus was monophyletic and included five species with the following cladistic relationship: (S. reevesii, (S. soemmerringii, (S. mikado, (S. humiae and S. ellioti)))). Using the TN genetic distance of cyt b, we inferred the divergence time of the five species according to putative molecular clock and found that values were largely in agreement with the geological scenarios. The origin and speciation processes of the studied group were inferred by combining molecular and biogeographical evidences.     

Pathogenicity of Haemoproteus meleagridis (Haemosporina: Haemoproteidae) in experimentally infected domestic turkeys

Sporozoite-induced experimental infections of Haemoproteus meleagridis produced a moderate to severe myositis and significant effects on weight gain and growth in domestic turkey poults. Pathological effects occurred in both low- and high-dose infections (4,400 and 57,500 sporozoites, respectively). Low-dose birds weighed significantly less than controls at 3 wk postinfection (PI) when peripheral parasitemia reached a peak and had significantly shorter tarsometatarsal lengths at both 1 and 3 wk PI. High-dose birds were significantly lighter and smaller than control and low-dose birds throughout the course of the 8-wk study. Infected birds were not anemic in spite of high parasitemias that often exceeded 50% of circulating erythrocytes. The most serious pathological effects occurred prior to patency and were associated with development of megaloschizonts in skeletal muscle. Microscopic lesions in 4 high-dose birds that died between 19 and 22 days PI were characteristic of a severe, acute hemorrhagic myositis. Megaloschizonts were surrounded by a hemorrhagic inflammatory infiltrate composed of macrophages, heterophils, giant cells, and red blood cells. Muscle fibers adjacent to megaloschizonts were swollen, hyaline, and contained prominent calcium deposits. Other observations included enlargement of the spleen, deposition of pigment in macrophages of the lung and spleen, and secondary bacterial and fungal infections in the intestine and lungs. Necrotic and calcified muscle fibers and degenerating megaloschizonts were still present at 8 wk PI when the experiment ended. Our results demonstrated significant pathological changes in H. meleagridis-infected domestic turkeys that were associated primarily with preerythrocytic stages of development.     

Two new species of Haemoproteus Kruse, 1890 (Haemosporida,Haemoproteidae) from European birds,with emphasis on DNA barcoding for detection of haemosporidians in wildlife

Two new species of Haemoproteus Kruse, 1890 (Haemosporida, Haemoproteidae) are described: Haemoproteus (Parahaemoproteus) homovelans n. sp. from Grey-faced Woodpecker, Picus canus Gmelin, and Haemoproteus (Parahaemoproteus) concavocentralis n. sp. recorded in Hawfinch, Coccothraustes coccothraustes (Linnaeus), both sampled in Bulgaria. The morphology of the gametocytes and their host-cells are described and mitochondrial cytochrome b (cyt b) gene sequences are generated. Haemoproteus homovelans possesses circumnuclear gametocytes lacking volutin granules. This parasite is particularly similar to Haemoproteus velans Coatney & Roudabush, 1937 also possessing circumnuclear gametocytes that are, however, overfilled with volutin. Haemoproteus concavocentralis can be readily distinguished from all described avian haemoproteids due to the presence of an unfilled concave space between the central part of advanced gametocytes and erythrocyte nucleus. Bayesian phylogenetic analyses of 40 haemosporidian cyt b lineages showed close relationships of H. concavocentralis (hHAWF2) with a group of Haemoproteus spp. possessing gametocytes that are pale-stained with Giemsa. The lineage hPICAN02 of H. homovelans clustered with parasites infecting non-passerine birds. Phylogenetic analyses support the current subgeneric classification of the avian haemoproteids and suggest that cyt b lineage hPIPUB01 (GenBank EU254552) has been incorrectly assigned to Haemoproteus picae Coatney & Roudabush, 1937, a common parasite of corvid birds (Passeriformes). This study emphasises the importance of combining molecular techniques and light microscopy in the identification and field studies of avian haemosporidian parasites. Future development of barcodes for molecular identification of haemoproteids will allow better diagnostics of these infections, particularly in veterinary studies addressing insufficiently investigated tissue pathology caused by these parasites.     

Haemoproteus gabaldoni n. sp. (Apicomplexa: Haemoproteidae) from the Muscovy duck Cairina moschata (Aves: Anatidae)

Haemoproteus gabaldoni n. sp. is described from the Muscovy duck Cairina moschata from Caracas, Venezuela and is compared to H. greineri and H. nettionis which have been described previously from the Anatidae. The highly amoeboid outline, volutin granules and small number of pigment granules of H. gabaldoni serve to readily separate this species from the other two.