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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.

Animals↗

Leucocytozoon hamiltoni n. sp. (Haemosporida, Leucocytozoidae) from the Bukharan great tit Parus bokharensis.

A new species of Leucocytozoon with gametocytes in fusiform host cells is described from the Bukharan great tit Parus bokharensis. It represents the first leucocytozoid in which the host cell nucleus is split into 2 more or less symmetrical portions, each located at an end of the host cell within its elongated fusiform processes. This species appears to be restricted with respect to geography and host.

Animals↗

Complete development of three species of Haemoproteus (Haemosporida, Haemoproteidae) in the biting midge Culicoides impunctatus (Diptera, Ceratopogonidae).

Development of Haemoproteus balmorali, H. dolniki, and H. tartakovskyi was followed in experimentally infected biting midges Culicoides impunctatus on the Curonian Spit in the Baltic Sea. Wild-caught flies were allowed to take blood meals on naturally infected spotted flycatchers Muscicapa striata, chaffinches Fringilla coelebs, or crossbills Loxia curvirostra harboring mature gametocytes of these parasites. The engorged biting midges were collected, held at 14-18 C, and dissected daily. Mature ookinetes of H. balmorali, H. dolniki, and H. tartakovskyi were numerous in the midgut content 36 hr postinfection (PI). Oocysts were first seen in the midgut wall 3 days PI. They were numerous in the midgut on the fourth day PI. Sporozoites were seen in salivary glands 5 days PI. The percentage of experimentally infected biting midges with sporozoites of H. balmorali was 36.1%: 8.8% with H. dolniki and 31.2% with H. tartakovskyi. Culicoides impunctatus is likely to be an important vector of Haemoproteus spp. in Europe. All investigated species of parasites can be distinguished on the basis of morphology or size (or both) of their vector stages. Morphological features of the ookinetes, oocysts, and sporozoites should be given more prominence in the description of new species of hemoproteids.

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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.

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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.

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Prior blood feeding effects on susceptibility of Anopheles gambiae (Diptera: Culicidae) to infection with cultured Plasmodium falciparum (Haemosporida: Plasmodiidae).

We examined the relative susceptibilities of Anopheles gambiae Giles of different physiological ages to infection with cultured Plasmodium falciparum (Welch). Cohorts of mosquitoes were divided into three groups; one was fed uninfected blood on day 3 after emergence (i.e., one prior blood meal); another on days 3 and 7 after emergence (i.e., two prior blood meals); and a control group was maintained on sucrose. On days 10 to 12 after emergence, mosquitoes were fed human blood containing P. falciparum gametocytes. Prior blood feeding accelerated digestion of the infective blood meals and subtly altered susceptibility to infection with P. falciparum. When gametocyte cultures were highly fertile, all experimental groups were equally susceptible to infection. However, when gametocyte fertility was low, accelerated digestion had a detrimental effect on the transition of ookinetes to oocysts. Accelerated digestion may raise the threshold density of ookinetes required for the successful conversion of ookinetes to oocysts.

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Exflagellation responses of cultured Plasmodium falciparum (Haemosporida: Plasmodiidae) gametocytes to human sera and midguts of anopheline mosquitoes (Diptera: Culicidae).

The process of exflagellation was quantified for cultured Plasmodium falciparum gametocytes exposed to human sera and midgut homogenates from six vector species of Anopheles mosquitoes. Neither serum factors related to malaria exposure nor factors in the midguts of taxonomically diverse anophelines had significant inhibitory effects on the exflagellation of P. falciparum microgametocytes. Therefore, differences in vector competence among anopheline species most likely are caused by vector-parasite interactions occurring after microgametogenesis.

Adult↗

Rheumatoid factor-like IgM in Plasmodium berghei (Apicomplexa: Haemosporida) infections of BALB/c mice.

Groups of female BALB/c mice infected by intravenous injection with 50 erythrocytes containing Plasmodium berghei Vincke et Lips, 1948 were sacrificed on days 3 through 12 after infection. Rheumatoid factor-like IgM (RF-IgM) and parasite-specific IgG levels were determined by enzyme-linked immunosorbent assay in serum specimens and in culture medium removed from spleen cell cultures established at sacrifice. All four mouse IgG subisotypes were recognized by RF-IgM molecules induced by Plasmodium berghei infection, and in this regard, the parasite-induced RF-IgM response resembled that induced by lipopolysaccharide polyclonal activation. Plasmodium berghei infection resulted in a biphasic RF-IgM response, with infected animals demonstrating significantly increased levels of RF-IgM early in the infection and significantly decreased levels late in the infection, compared to uninfected control mice. The decreased levels of RF-IgM observed late in infection correlated with increasing parasitaemia levels, and were primarily due to a decrease in RF-IgM specific for mouse IgG2a. Late infection levels of RF-IgM specific for IgGI, IgG2b, and IgG3 were not significantly different from those of control animals.

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Testosterone levels and gular pouch coloration in courting magnificent frigatebird (Fregata magnificens): variation with age-class, visited status and blood parasite infection.

Male magnificent frigatebirds (Fregata magnificens) possess a seasonally expressed skin ornament, namely the red and inflatable gular pouch, and are, therefore, a convenient model for the study of some theories related to the evolution of possible testosterone-dependent sexual skin coloration. Here we report the findings of a study performed over four consecutive mating seasons in the Mexican national park Isla Isabel. We investigated differences in testosterone level and gular pouch coloration in courting males in relation to the categories: age-class, visited status and blood parasite infection. Gular pouch color saturation increased with age-class. Investigated frigatebirds were infected only with Haemoproteus iwa (Haemosporida, Haemoproteidae), with an overall prevalence infection of 15.5%. Prevalence of the infection increased with birds' age-class. Testosterone levels were significantly higher in infected males, who also had lighter colored gular pouches. In non-infected males, those visited by a female had higher testosterone levels than non-visited males. Gular pouch lightness and redness were negatively correlated but only redness in non-visited non-infected males was positively correlated with testosterone levels. Gular pouch saturation in visited and infected males was positively correlated with body mass, which also increased with age-class. Mated males had lower testosterone levels and lighter, less red and saturated gular pouch coloration than courting males. In summary, we found that coloration of the male skin ornament could reflect age-class (saturation), parasite infection (lightness) and mated status (all), together with indications of condition and testosterone dependency of ornament expression.

Aging↗

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).

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Nested cytochrome b polymerase chain reaction diagnostics underestimate mixed infections of avian blood haemosporidian parasites: microscopy is still essential.

Numerous polymerase chain reaction (PCR)-based methods have been developed and used increasingly to screen vertebrate blood samples for the diagnosis of haemosporidian blood parasites (Sporozoa, Haemosporida), but a rigorous evaluation of the sensitivity of these methods for detecting mixed infections of different haemosporidian species belonging to the same and different genera and subgenera is lacking. This study links the information obtained by nested cytochrome b PCR and traditional microscopy in determining mixed haemosporidian infections in naturally infected birds. Samples from 83 individual passerine birds with single infections of Haemoproteus or Plasmodium spp., as determined by mitochondrial DNA amplification, also were investigated by microscopic examination of stained blood films. Thirty-six samples (43%) were found to harbor mixed Haemoproteus, or Plasmodium spp. infections, or both. Thus, the PCR assays alone underestimate the occurrence of mixed infections of haemosporidian parasites in naturally infected birds. To determine the true species composition of the haemosporidians in each individual host, PCR diagnostics need to be improved. Specific primers for Haemoproteus spp. and Plasmodium spp. should be developed. Ideally, a combination of the approaches of both microscopy and PCR-based methods is recommended for this purpose.

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Integrated mapping, chromosomal sequencing and sequence analysis of Cryptosporidium parvum.

The apicomplexan Cryptosporidium parvum is one of the most prevalent protozoan parasites of humans. We report the physical mapping of the genome of the Iowa isolate, sequencing and analysis of chromosome 6, and approximately 0.9 Mbp of sequence sampled from the remainder of the genome. To construct a robust physical map, we devised a novel and general strategy, enabling accurate placement of clones regardless of clone artefacts. Analysis reveals a compact genome, unusually rich in membrane proteins. As in Plasmodium falciparum, the mean size of the predicted proteins is larger than that in other sequenced eukaryotes. We find several predicted proteins of interest as potential therapeutic targets, including one exhibiting similarity to the chloroquine resistance protein of Plasmodium. Coding sequence analysis argues against the conventional phylogenetic position of Cryptosporidium and supports an earlier suggestion that this genus arose from an early branching within the Apicomplexa. In agreement with this, we find no significant synteny and surprisingly little protein similarity with Plasmodium. Finally, we find two unusual and abundant repeats throughout the genome. Among sequenced genomes, one motif is abundant only in C. parvum, whereas the other is shared with (but has previously gone unnoticed in) all known genomes of the Coccidia and Haemosporida. These motifs appear to be unique in their structure, distribution and sequences.

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[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.

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The fine structure of Garnia gonadati and its association with the host cell.

Most of the studies on the fine structure of protozoa of the Apicomplexa group have been carried out with members of the ToxoPlasma, Eimeria, and Plasmodium genera. In the present study we analyzed the fine structure of Garnia gonadoti parasitizing the red blood cells of the Amazonian reptile Gonatodes humeralis (Reptilia; Lacertilia). Transmission electron microscopy of thin sections showed that G. gonadoti presented all structures characteristic of the group, including the apicoplast. However, four special features were observed: (1) absence of the hemozoin (malarial) pigment; (2) a group of microtubules associated with the mitochondrion; (3) a vacuole containing electron-dense material, which resembled the acidocalcisome described in trypanosomatids; and (4) a special array of the host-cell endoplasmic reticulum around the parasitophorous vacuole.

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Factors affecting the relapse of Haemoproteus belopolskyi infections and the parasitaemia of Trypanosoma spp. in a naturally infected European songbird, the blackcap, Sylvia atricapilla.

The effects of photoperiod and stress on the relapse of Haemoproteus belopolskyi infections and the parasitaemia of Trypanosoma spp. were studied in naturally infected blackcaps, Sylvia atricapilla. Twenty-one birds (12 infected with H. belopolskyi, 5 with Trypanosoma spp.) were kept indoors and investigated by microscopic examination of stained blood films. All infections were latent before the experiments started on 7 February 2002. The relapse of H. belopolskyi and the parasitaemia of Trypanosoma spp. after the latent stage of infection were induced by increased day length and stress due to a change of room. At 44 days after the beginning of the experiment, all latent infections were patent in the experimental birds, while no parasites were recorded in the control birds. The relapse of avian Haemoproteus spp. infection and of the parasitaemia of avian Trypanosoma spp. may be related to gonadal hormones, corticosterone and melatonin.

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Leucocytozoonosis in the Israeli sparrow, Passer domesticus biblicus Hartert 1904.

Among the 91 house sparrows (Passer domesticus biblicus Hartert, 1904) examined and caught in the Jordan valley, Israel, 79% were found to be infected with Leucocytozoon fringillinarum Woodcock 1910. In the coastal plain of Israel (South of Tel Aviv), Leucocytozoon infection was found in only 3 out of 43 examined sparrows. In the birds examined, Leucocytozoon gametocytes were present, often in large numbers, in the circulating blood of the visceral organs, whereas they were only sporadic or even absent in the peripheral blood. Gametocytes were seen in the brain capillaries in only a few birds. Only one of the heavily infected sparrows was anemic. Leucocytozoon merozoites were present in the liver and kidneys in only a few infected birds. Merogonic infections did not induce any severe pathological changes, while the gametocyte congestion caused dilation of the blood vessels and sinuses. Tissue damage by the gametocyte parasitemia was most evident in the liver and kidneys. Leucocyte infiltration developed alongside the affected vessels; diffuse necrosis developed in the infiltrated areas. In the kidneys, many tubules were degenerated. Leucocytozoon gametocyte infection in sparrows is unique in that it appears to be confined, for most of its duration, to the visceral circulation, resulting in clinical consequences. Geographically, it is confined to habitats presumably supporting vectors.

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