Next-generation sequencing reveals cryptic mtDNA diversity of Plasmodium relictum in the Hawaiian Islands.

Next-generation 454 sequencing techniques were used to re-examine diversity of mitochondrial cytochrome b lineages of avian malaria (Plasmodium relictum) in Hawaii. We document a minimum of 23 variant lineages of the parasite based on single nucleotide transitional changes, in addition to the previously reported single lineage (GRW4). A new, publicly available portal (Integroomer) was developed for initial parsing of 454 datasets. Mean variant prevalence and frequency was higher in low elevation Hawaii Amakihi (Hemignathus virens) with Avipoxvirus-like lesions (P = 0·001), suggesting that the variants may be biologically distinct. By contrast, variant prevalence and frequency did not differ significantly among mid-elevation Apapane (Himatione sanguinea) with or without lesions (P = 0·691). The low frequency and the lack of detection of variants independent of GRW4 suggest that multiple independent introductions of P. relictum to Hawaii are unlikely. Multiple variants may have been introduced in heteroplasmy with GRW4 or exist within the tandem repeat structure of the mitochondrial genome. The discovery of multiple mitochondrial lineages of P. relictum in Hawaii provides a measure of genetic diversity within a geographically isolated population of this parasite and suggests the origins and evolution of parasite diversity may be more complicated than previously recognized.

Phagocytosis of liposomes by macrophages: intracellular fate of liposomal malaria antigen.

Liposomes containing a synthetic recombinant protein were phagocytosed by macrophages, and the internalized protein was recycled to the cell surfaces where it was detected by enzyme-linked immunosorbent assay. The transit time of the liposome-encapsulated protein from initial phagocytosis of liposomes to appearance of protein on the surfaces of macrophages was determined by pulse-chase experiments. The macrophages were pulsed with liposomes containing protein and chased with empty liposomes, and vice versa. The amount and rate of protein antigen expression at the cell surfaces depended on the quantity of encapsulated protein ingested by the macrophages. Although liposomes were rapidly taken up by macrophages, the liposome-encapsulated protein was antigenically expressed for a prolonged period (at least 24 h) on the cell surface. Liposomes were visualized inside vacuoles in the macrophages by immunogold electron microscopy. The liposomes accumulated along the peripheries of the vacuoles and many of them apparently remained intact for a long time (greater than 6 h). However, nonliposomal free protein was also detected in the cytoplasm surrounding these vacuoles, and it was concluded that the free protein in the cytoplasm was probably en route to the macrophage surface. Exposure of the cells to ammonium chloride did not inhibit the appearance of liposomal antigenic epitopes on the cell surface, and this suggests that expression of the liposomal antigenic epitopes at the surface was not a pH-sensitive phenomenon. There was no significant effect of a liposomal adjuvant, lipid A, on the rate or extent of surface expression of the liposomal protein.

Ultrastructural localization of erythrocyte cytoskeletal and integral membrane proteins in Plasmodium falciparum-infected erythrocytes.

The distributions of ankyrin, spectrin, band 3, and glycophorin A were examined in Plasmodium falciparum-infected erythrocytes by immunoelectron microscopy to determine whether movement of parasite proteins and membrane vesicles between the parasitophorous vacuole membrane and erythrocyte surface membrane involves internalization of host membrane skeleton proteins. Monospecific rabbit antisera to spectrin, band 3 and ankyrin and a mouse monoclonal antibody to glycophorin A reacted with these erythrocyte proteins in infected and uninfected human erythrocytes by immunoblotting. Cross-reacting malarial proteins were not detected. The rabbit sera also failed to immunoprecipitate [3H]isoleucine labeled malarial proteins from Triton X-100 and sodium dodecyl sulfate (SDS) extracts of infected erythrocytes. These three antibodies as well as the monoclonal antibody to glycophorin A bound to the membrane skeleton of infected and uninfected erythrocytes. The parasitophorous vacuole membrane was devoid of bound antibody, a result indicating that this membrane contains little, if any, of these host membrane proteins. With ring-, trophozoite- and schizont-infected erythrocytes, spectrin, band 3 and glycophorin A were absent from intracellular membranes including Maurer's clefts and other vesicles in the erythrocyte cytoplasm. In contrast, Maurer's clefts were specifically labeled by anti-ankyrin antibody. There was a slight, corresponding decrease in labeling of the membrane skeleton of infected erythrocytes. A second, morphologically distinct population of circular, vesicle-like membranes in the erythrocyte cytoplasm was not labeled with anti-ankyrin antibody. We conclude that membrane movement between the host erythrocyte surface membrane and parasitophorous vacuole membrane involves preferential sorting of ankyrin into a subpopulation of cytoplasmic membranes.

Characterization of a sporozoite antigen common to Plasmodium falciparum and Plasmodium berghei.

Previous studies demonstrated that immunization with Plasmodium falciparum sporozoites protected mice against Plasmodium berghei sporozoite infection and that this cross-protection was mediated, at least in part, by anti-sporozoite antibody. The experiments presented in this report show that serum and monoclonal antibodies derived from these protected mice identify a novel 42/54-kDa antigen (designated Circumsporozoite Protein 2 or CSP-2) in both P. falciparum and P. berghei sporozoites. Anti-CSP-2 monoclonal antibody blocks invasion of P. falciparum and P. berghei sporozoites into hepatoma cells in vitro and binds the cell surface of sporozoites. Passive transfer of anti-CSP-2 monoclonal antibody protected mice from P. berghei sporozoite infection. Therefore, CSP-2 appears to play a role in the cross-protective immune response observed.

Induction of Plasmodium falciparum sporozoite-neutralizing antibodies upon vaccination with recombinant Pfs16 vaccinia virus and/or recombinant Pfs16 protein produced in yeast.

Pfs16 is a sexual stage/sporozoite-specific antigen of Plasmodium falciparum and is a potential candidate for a sporozoite-neutralizing vaccine. To obtain more information on the function of Pfs16 and to investigate its role during transmission and hepatocyte invasion, immunization experiments were performed with both a Pfs16-specific recombinant vaccinia virus and virus-like particles produced in yeast composed of the hepatitis B surface antigen (HBsAg) and antigen Pfs16 fused to HBsAg. Upon transformation of yeast cells, harbouring a genomic copy of the HBsAg gene, with a plasmid carrying the fusion gene Pfs16-HBsAg (Pfs16-S) virus-like hybrid particles composed of HBsAg and Pfs16-S were formed of a size similar to those present in human sera after infection with the hepatitis B virus. Cells infected with recombinant Pfs16 vaccinia virus synthesized a polypeptide of approx. 16 kDa that reacted with a Pfs16-specific polyclonal antibody. Animals vaccinated with the yeast hybrid particles and/or recombinant vaccinia virus both produced Pfs16-specific antibodies. These antibodies showed no transmission-blocking activity, but they efficiently diminished or abolished in vitro invasion of sporozoites into human hepatoma cells (HepG2-A16) and primary human hepatocytes.

Non-CS pre-erythrocytic protective antigens.

Three novel non-CS antigens have been identified on P. falciparum and P. berghei sporozoites and exoerythrocytic parasites. CSP-2 is a sporozoite surface protein common to P. falciparum and P. berghei that elicits antibody-mediated protection, and is also found within P. berghei EE parasites. LSA is a P. falciparum EE-specific antigen localized within the parasitophorous vacuole. LSA-2 is a P. berghei EE-specific antigen, localized on the parasitophorous vacuole membrane, that protected mice to P. berghei sporozoite challenge, and elicited cytotoxic T cells that killed P. berghei EE parasites in vitro.

Localization of a 230-kD parasitophorous vacuole membrane antigen of Plasmodium berghei exoerythrocytic schizonts (LSA-2) by immunoelectron and confocal laser scanning microscopy.

Using antiserum to a 230-kD parasitophorous vacuole membrane (PVM) antigen of Plasmodium berghei exoerythrocytic schizonts as a specific probe for the PVM, we studied the three-dimensional structure of this membrane within infected host cells by immunoelectron microscopy and confocal laser scanning microscopy at 3, 4, and 50 hr after sporozoite invasion. Fluorescent label was not detected at 3 hr, but was associated with the cytoplasm of 24-hr-old exoerythrocytic parasites. Specific labeling of the PVM was not observed by immunoelectron microscopy until 50 hr, when numerous vesicles and finger-like projections of the PVM were found in the cytoplasm of infected host cells. Labeled vesicles were often isolated and located at the periphery of the infected hepatocyte. Confocal microscopy demonstrated that these vesicles formed discontinuous chains that extended from 3-10 microns away from the parasite. These structures appear to be similar to the membranous clefts of Plasmodium-infected erythrocytes, and may be important in the movement of host or parasite proteins within infected hepatocytes.

Localization of circumsporozoite antigen in exoerythrocytic schizonts of Plasmodium cynomolgi.

We used colloidal gold probes and post-embedding immunoelectron microscopy to localize circumsporozoite (CS) antigen in 5- and 8-day-old in vitro cultures of Plasmodium cynomolgi exoerythrocytic (EE) schizonts. Both small uninucleated and large multinucleated EE schizonts were found in 5-day-old cultures. A mouse monoclonal antibody to the repeat region of the P. cynomolgi CS protein densely labeled the plasma membrane and surface of 5-day-old EE schizonts as well as the surrounding parasitophorous vacuole membrane and space. Density of labeling decreased significantly as EE schizonts increased in size and maturity. Labeling of large, multinucleated 5-day-old schizonts was sparse and limited to the surface of EE schizonts and to small patches of electron dense material which were attached to the inner surface of the parasitophorous vacuole membrane. Mature 8-day-old EE schizonts with developing merozoites had little detectable labeling. CS antigen was not associated with internal structures within developing schizonts. Labeling was not observed in the host cell cytoplasm or on the surface of infected hepatocytes. These findings indicate that epitopes associated with the repeat region of the P. cynomolgi circumsporozoite protein are sequestered within infected host cells during EE development.

Expression of Plasmodium berghei circumsporozoite antigen on the surface of exoerythrocytic schizonts and merozoites.

The intracellular distribution of circumsporozoite (CS) antigen was traced by immunoelectron microscopy in cultures of Plasmodium berghei exoerythrocytic (EE) schizonts with monoclonal antibody (Mab) 3D11 to the immunodominant repeat region of the P. berghei CS protein. CS antigen was localized on the parasitophorous vacuole (PV) membrane and pellicular complex of recently invaded sporozoites and on electron-dense masses of sloughed CS antigen in the host cell cytoplasm. CS antigen persisted throughout the complete EE cycle of P. berghei on the surface of EE schizonts and was incorporated into the plasma membrane of budding EE merozoites. Erythrocytic merozoites were not labeled by Mab 3D11, indicating that these 2 populations of merozoites differ in antigenic composition. Significant internal labeling occurred in 50 hr EE schizonts in association with the limiting membranes of peripheral vesicles and short, tube-like structures attached to their outer surfaces. These vesicles contained an electron-dense flocculent material also present in the PV space. Association of CS antigen with the limiting membranes of these vesicles suggests that they either develop as endocytotic invaginations of the schizont plasma membrane or transport newly synthesized CS antigen from the endoplasmic reticulum and Golgi of developing EE schizonts to the parasite surface.

Strain specificity in the liver-stage development of Plasmodium falciparum in primary cultures of new world monkey hepatocytes.

Primary cultures of Aotus and Saimiri monkey hepatocytes were infected with sporozoites of the Plasmodium falciparum NF 54 strain from mosquitoes fed on gametocyte cultures, and with sporozoites of the P. falciparum Santa Lucia strain from mosquitoes fed on an infected Aotus monkey. After 4-8 days, one exoerythrocytic (EE) parasite per 30,000 sporozoites was detected in one of three experiments performed with the P. falciparum NF54 strain. However, numerous EE parasites were detected in Aotus and Saimiri cells infected with sporozoites of the P. falciparum Santa Lucia strain. At day 6, most of the parasites contained several hundred nuclei, and were morphologically similar to those previously described in vivo using light or electron microscopy. A monoclonal antibody directed against the repeat region of the circumsporozoite protein of P. falciparum labeled the plasma and parasitophorous vacuole membrane of five-day-old EE parasites by immunoelectron microscopy, thus supporting previous observations by immunofluorescence indicating that the CS protein persists throughout the EE development of P. falciparum. These results demonstrate that liver stages of P. falciparum can be obtained in Aotus and Saimiri monkey cells, they also suggest a parasite strain specificity for hepatocytes.

Stage-specific ultrastructural effects of desferrioxamine on Plasmodium falciparum in vitro.

Desferrioxamine (DFO) is an iron chelator that inhibits the in vitro and in vivo growth of rodent and human malarial parasites. Previous studies with this chelator have suggested that it might interfere with the intraerythrocytic growth of Plasmodium sp. by withholding iron from any of several essential iron-dependent parasite enzymes, including those involved in CO2 fixation, mitochondrial electron transport, pyrimidine synthesis, and the reduction of ribonucleotides for DNA synthesis. We studied the ultrastructural effects of DFO on synchronized cultures of P. falciparum to identify the specific site of action of this compound. Synchronized cultures of early rings or schizonts were exposed to 100 microM DFO for up to 48 hr, and fixed and processed at regular intervals for electron microscopy. Untreated cultures and cultures exposed to DFO saturated with Fe3+ were processed at the same time. When DFO was added to synchronized cultures containing early rings, parasites developed normally until the late trophozoite stage, when all growth ceased. Ultrastructural lesions included the breakdown of the nuclear envelope into small membranous fragments and progressive vacuolization of the nucleoplasm. Other organelles, including food vacuoles and mitochondria, were not affected. The addition of DFO to synchronized cultures of schizonts had similar effects on nuclei of early schizonts, but little or no effect on mature schizonts and segmenters. Erythrocyte invasion by merozoites proceeded in the presence of the chelator. These findings support the hypothesis that DFO acts specifically during the late trophozoite/early schizont stage of parasite maturation by preventing nuclear division, an effect consistent with inhibition of the iron-dependent enzyme ribonucleotide reductase.

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.

Isospora manchacensis n. sp., an intranuclear coccidian from the Louisiana ground skink, Scincella lateralis (Say, 1823) (Lacertilia: Scincidae).

Isospora manchacensis n. sp. is described from ground skinks, Scincella lateralis (Say, 1823) from Louisiana. Overall prevalence at 6 sites near Lake Ponchartrain was 43.1% (59/137) and ranged from 8% (1/13) to as high as 60% (6/10). Endogenous stages develop inside the nuclei of epithelial cells in the small intestine. Infected hypertrophic nuclei migrate from the basal lamina of the host cell to the luminal striated border. Oocysts in freshly passed fecal pellets usually contain a single contracted sporont that divides to form 2 sporoblasts. These undergo a brief pyramid stage followed by sporulation within 45-50 hr. Sporulated oocysts have a single-layered wall and measure 25.0 X 22.6 (20.0-28.9 X 18.6-26.0) micron. The lemon-shaped sporocysts measure 12.8 X 10.2 (11.1-15.2 X 9.0-11.0) micron and contain a Steida body, a spherical to oval substeida body, and a dispersed, granular sporocyst residuum. Prepatent periods in skinks fed 700 and 1,400 oocysts ranged from 24 to 32 days. Experimentally infected skinks produced large numbers of oocysts continuously during the 3-4 wk they were monitored after the onset of patency, but exhibited no signs of disease. Experimental doses of 200 oocysts failed to produce infections in skinks monitored for as long as 7 wk.

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.

Ultrastructural localization of Plasmodium falciparum circumsporozoite protein in newly invaded hepatoma cells.

The fate and disposition of the circumsporozoite (CS) protein of Plasmodium falciparum was investigated during hepatoma cell invasion with several sera raised against defined CS peptides, including both repeat and nonrepeat regions spanning approximately 60% of the P. falciparum CS gene product. Distribution of the protein, as revealed by immunoelectron microscopy, was limited to the surface of the sporozoite both before and after invasion. In particular, no CS protein antigen was detected in association with either the parasitophorous vacuole membrane or the host cell surface.

Ultrastructural localization of protective and nonprotective Plasmodium falciparum proteins using serum samples from vaccinated Aotus monkeys.

Postembedding immunoelectron microscopy, using pooled serum samples from a recent vaccination experiment involving Aotus monkeys, was used to localize immune targets in Plasmodium falciparum-infected erythrocytes and free merozoites. Serum samples from Aotus monkeys, protected completely by immunization with the P. falciparum merozoite surface coat precursor protein, identified immune targets on the surface of free and intracellular merozoites as well as the cytoplasm, plasma membrane, and parasitophorous vacuole membrane of immature schizonts. Serum samples from unprotected monkeys, which had been immunized with a complex of 143-kDa, 132-kDa, and 102-kDa polypeptides reacted specifically with the rhoptries of immature schizonts and mature merozoites.

Myopathy associated with megaloschizonts of Haemoproteus meleagridis in a wild turkey from Florida.

Necropsy of an emaciated adult wild turkey (Meleagris gallopavo osceola) that died in captivity soon after capture revealed numerous macroscopic 1-2 mm white cysts in the pectoral muscles. Microscopic, aseptate protozoan megaloschizonts, 50-150 microns in diameter, corresponded to the cysts in histological sections. The megaloschizonts were surrounded by a thick, hyaline wall and packed with spherical merozoites less than 1 micron in diameter. Muscle fibers surrounding most of the megaloschizonts exhibited early signs of dystrophic calcification. The fibers were swollen, pale and hyaline and contained scattered basophilic granules. The megaloschizonts were morphologically distinct from sarcocysts of Sarcocystis sp. and Besnoitia sp. and the thin-walled tissue cysts of Toxoplasma gondii. They were identical in structure and host reaction to the second-generation megaloschizonts of Haemoproteus meleagridis, reported previously from experimentally infected domestic turkeys. While the precise cause of death of the wild turkey could not be determined, the most prominent lesions were associated with the numerous intramuscular megaloschizonts.

Sarcocystis sp. in wading birds (Ciconiiformes) from Florida.

Sarcocysts were found in striated muscle of 21 adult wading birds among 145 examined grossly and 70 examined histologically (calculated prevalence = 24%), and in none of 332 immature wading birds examined from Florida (USA). Six of 12 species of ciconiforms were infected (Ardea herodias, Casmerodius albus, Egretta caerulea, Nyctanassa violacea, Butorides striatus, Eudocimus albus). Cysts were filamentous, usually extended the entire length of the muscle fiber, and were visible grossly in 33% of the positive cases. We concluded from ultrastructural examination of cysts that the same species of Sarcocystis may occur in all species of wading birds in Florida; however, two cyst diameters were noted that appeared to differ in their distribution by host species.

Serological responses and immunity to superinfection with avian malaria in experimentally-infected Hawaii amakihi.

Six of seven Hawaii Amakihi (Hemignathus virens) with chronic malarial infections had no increases in peripheral parasitemia, declines in food consumption, or loss of body weight when rechallenged with the homologous isolate of Plasmodium relictum 61 to 62 days after initial infection. Five uninfected control amakihi exposed at the same time to infective mosquito bites developed acute infections with high parasitemias. Reductions in food consumption and loss of body weight occurred in all control birds and three of these individuals eventually died. When surviving birds were rechallenged >2 yr later with either the same parasite isolate or an isolate of P. relictum collected on the island of Kauai, all individuals were immune to superinfection. Chronically infected birds developed antibodies to a common suite of malarial antigens ranging in size from 22 to 170 kDa that were detectable as early as 8 days post infection on immunoblots of SDS-polyacrylamide gels. Antibodies to this suite of malarial antigens persisted as long as 1,248 days after initial infection and were consistently detectable at times when parasites were not easily found by microscopy on Giemsa-stained blood smears. The immunoblotting method that is described here appears to be an effective technique for identifying birds with chronic, low-intensity malarial infections when circulating parasites are not easily detectable by microscopy. Hawaiian honeycreepers that are capable of recovering from acute infections develop concomitant immunity to superinfection, making them functionally immune in areas where malaria transmission has become endemic.