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.     

Pre-erythrocytic development and associated host responses to Haemoproteus meleagridis (Haemosporina: Haemoproteidae) in experimentally infected domestic turkeys

Two generations of pre-erythrocytic schizogony occurred in skeletal and cardiac muscle of domestic turkeys infected with sporozoites of Haemoproteus meleagridis. First generation schizonts reached maturity approximately five days post-inoculation (DPI) and developed in capillary endothelial cells and myofibroblasts. The schizonts ranged from 12 to 20 microns in diameter and produced long (5-6 microns), slender merozoites. Early second generation schizonts were first detected in capillary endothelial cells between 5 and 8 DPI. They were cylindrical and ranged in size from 5 to 8 microns in diameter and up to 28 microns in length. Second generation schizonts which reached maturity by 17 DPI were surrounded by a thick, hyaline wall and were packed with numerous spherical merozoites less than 1 micron in diameter. Mature megaloschizonts were fusiform, ranged from 30 to 113 microns in diameter, and extended as much as 465 microns along the long axis of muscle fibers. Merozoites developed as buds from cytomeres that formed between 8 and 14 DPI. Infected turkeys developed a moderate to severe myositis within 5 DPI and were lame in one or both legs. The myositis was associated with the necrosis of scattered groups of muscle fibers. Muscle fibers surrounding mature megaloschizonts were swollen and hyaline. Megaloschizonts were surrounded occasionally by fibroblasts and infiltrates of mononuclear cells. The morphology and site of development of mature megaloschizonts of Haemoproteus meleagridis are contrasted with those of other avian haemosporidians.     

Pre-Erythrocytic Development and Associated Host Responses to Haemoproteus meleagridis (Haemosporina: Haemoproteidae) in Experimentally Infected Domestic Turkeys12

ABSTRACT. Two generations of pre-erythrocytic schizogony occurred in skeletal and cardiac muscle of domestic turkeys infected with sporozoites of Haemoproteus meleagridis. First generation schizonts reached maturity approximately five days post-inoculation (DPI) and developed in capillary endothelial cells and myofibroblasts. The schizonts ranged from 12 to 20 μm in diameter and produced long (5–6 μm), slender merozoites. Early second generation schizonts were first detected in capillary endothelial cells between 5 and 8 DPI. They were cylindrical and ranged in size from 5 to 8 μm in diameter and up to 28 μm in length. Second generation schizonts which reached maturity by 17 DPI were surrounded by a thick, hyaline wall and were packed with numerous spherical merozoites less than 1 μm in diameter. Mature megaloschizonts were fusiform, ranged from 30 to 113 μm in diameter, and extended as much as 465 μm along the long axis of muscle fibers. Merozoites developed as buds from cytomeres that formed between 8 and 14 DPI. Infected turkeys developed a moderate to severe myositis within 5 DPI and were lame in one or both legs. The myositis was associated with the necrosis of scattered groups of muscle fibers. Muscle fibers surrounding mature megaloschizonts were swollen and hyaline. Megaloschizonts were surrounded occasionally by fibroblasts and infiltrates of mononuclear cells. The morphology and site of development of mature megaloschizonts of Haemoproteus meleagridis are contrasted with those of other avian haemosporidians.     

Epizootiology of Haemoproteus danilewskyi (Haemosporina: Haemoproteidae) in blue jays (Cyanocitta cristata) in southcentral Florida

Prevalence and density of Haemoproteus danilewskyi was studied in a population of free-ranging blue jays (Cyanocitta cristata) in southcentral Florida (USA) from May 1992 to December 1995. Prevalence of infection was 27% for data combined over years, seasons, ages, and sexes. Prevalence did not vary between sexes or among years, but increased with age and varied with season, being highest in June-July and lowest in November-January. Parasite density did not vary between sexes or among seasons, but was higher in younger birds when controlling for season. To determine periods of natural transmission, seasonal patterns of infection were compared with previous month abundance of the biting fly vectors. Mean monthly prevalence of H. danilewskyi in older jays was positively correlated with previous month abundance of Culicoides edeni and C. arboricola, both capable of sporogonic development of H. danilewskyi.     

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.     

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.     

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.     

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.     

Leucocytozoon toddi and Haemoproteus tinnunculi (Protozoa: Haemosporina) in the Chimango caracara (Milvago chimango) in southern Chile.

Two species of blood protozoans were identified from blood smears collected from 15 specimens of the Chimango caracara (Milvago chimango) on Isla Grande de Chiloé in southern Chile. These included Leucocytozoon toddi in 13 birds, including all 5 of the 4-6 week old nestlings examined, and 8 of the subadults or adults. One of the nestlings also had a dual infection of L. toddi and Haemoproteus tinnunculi. These are the first reports of blood parasites from M. chimango.     

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.     

Haemoproteus palumbis sp. nov. (Sporozoa, Haemosporina) of the English Wood-Pigeon Columba p. palumbus

SYNOPSIS. Haemoproteus palumbis sp. nov. is described from the English wood-pigeon, Columba p. palumbus. Its pigmented gametocytes inhabit erythrocytes and resemble those of H. columbae (in C. livia ) but may be slightly longer and narrower. It is characterized by having oval or slightly lobed, not elongate, schizonts in endothelial cells of lung and heart, a prepatent period of 14 days, and a sporogonic cycle in the hippoboscid fly Ornithomya avicularia lasting 6 1/2–7 days. H. palumbis sp. nov. cannot infect C. livia but can infect a small proportion of individuals of Pseudolynchia canariensis , the vector of H. columbae.     

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.     

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.