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.

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.

Pathogenicity of avian malaria in experimentally-infected Hawaii Amakihi.

The introduction of avian malaria (Plasmodium relictum) and mosquitoes (Culex quinquefasciatus) to the Hawaiian Islands (USA) is believed to have played a major role in the decline and extinction of native Hawaiian honeycreepers (Drepanidinae). This introduced disease is thought to be one of the primary factors limiting recovery of honeycreepers at elevations below 1,200 m where native forest habitats are still relatively intact. One of the few remaining species of honeycreepers with a wide elevational distribution is the Hawaii Amakihi (Hernignathus virens). We measured morbidity and mortality in experimentally-infected Hawaii Amakihi that were captured in a high elevation, xeric habitat that is above the current range of the mosquito vector. Mortality among amakihi exposed to a single infective mosquito bite was 65% (13/20). All infected birds had significant declines in food consumption and a corresponding loss in body weight over the 60 day course of the experiment. Gross and microscopic lesions in birds that succumbed to malaria included enlargement and discoloration of the spleen and liver and parasitemias as high as 50% of circulating erythrocytes. Mortality in experimentally-infected amakihi was similar to that observed in Apapane (Himnatione sanguinea) and lower than that observed in Iiwi (Vestiaria coccinea) infected under similar conditions with the same parasite isolate. We conclude that the current elevational and geographic distribution of Hawaiian honeycreepers is determined by relative susceptibility to avian malaria.

Characterization of poxviruses from forest birds in Hawaii.

Two strains of avian pox viruses were isolated from cutaneous lesions in Hawaiian crows (Corvus hawaiiensis) examined in 1994 and a third from a biopsy obtained in 1992 from an infected bird of the Apapane species (Himatione sanguinea) by inoculation of the chorioallantoic membranes (CAM) of developing chicken embryos. The resulting proliferative CAM lesions contained eosinophilic cytoplasmic inclusion bodies characteristic of pox virus infection. The pathogenicity of these three viruses in domestic chickens was mild as evidenced by the development of relatively minor lesions of short duration at the sites of inoculation. Their virulence in this host was similar to that of a fowlpox virus (FPV) vaccine strain and contrasted greatly with the ability of two field strains of FPV to produce extensive proliferative lesions. One of the Hawaiian crow pox virus isolates as well as the one originating from the Apapane species could be propagated in two secondary avian cell lines, QT-35 and LMH. A comparison of the restriction fragment length polymorphisms (RFLP) of the genomes of the two cell line-adapted viruses, generated by EcoRI digestion, revealed a limited degree of similarity. Moreover, neither profile was comparable to those of the two field isolates of FPV, which were almost indistinguishable from each other. Thus, based on the genetic distinctness of the two Hawaiian bird viruses, they appear to represent different strains of avipoxvirus.

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.

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.

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.

Morphology, prevalence, and distribution of Sarcocystis spp. in white-tailed deer (Odocoileus virginianus) from Florida.

Three morphologically distinct types of sarcocysts (I, II, and III) were identified by light microscopy in tongues from 403 white-tailed deer (Odocoileus virginianus) collected in Florida (USA) over a 7-yr-period. Based on electron microscopy of representative examples of these sarcocysts, there were four distinctive wall structures. We concluded that one of these (Type I) was representative of Sarcocystis odocoileocanis and another (Type III) was representative of an unnamed species previously described from white-tailed deer in Montana. Type II could be divided further into two forms (IIA and IIB) that may represent two underscribed species or developmental stages of the same species. Sarcocystis odoi, another previously recognized sarcosporidian from white-tailed deer, was not found. Sarcocysts of Types I and II were distributed nonrandomly in tongue muscle, being more common in the basal portion, whereas Type III was distributed randomly throughout the tongue. Single infections (one of the three types) accounted for 63% of the infected deer, while double infections occurred in 28% and triple infections in 4%. Types I and II were found in deer throughout the state, but Type III occurred only in deer from southern Florida. In 1988 and 89, the statewide prevalences for Types I, II, and III were 57, 20, and 6%, respectively. Prevalences of Type I ranged from 94% in the panhandle region (northern Florida) to 34% in the southern part of the state. Prevalences of all three types increased with age.

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.

Sarcocysts in the Florida bobcat (Felis rufus floridanus).

Sarcocysts were found in the tongue, diaphragm, heart, intestinal tunica muscularis, and skeletal muscle of bobcats (Felis rufus floridanus) collected in Florida (USA). The tongue was found to be the best indicator tissue for sarcocysts (P less than 0.005). Thirty of 60 bobcats screened were found to contain sarcocysts in at least one of the muscle tissues examined. Of the positive bobcats, 28 of 28 tongues contained sarcocysts, while only 10 of 27 (37%), and 8 of 26 (31%) contained sarcocysts in the diaphragm or cardiac muscle, respectively. Although immune suppression has been suggested as a possible reason for formation of sarcocysts in some carnivores, no such correlation was evident in the bobcats. Comparisons of prey species taken by the panther and bobcat, and overlap of geographical range by the two species leave questions as to the source of infection, and the species of Sarcocystis that is infecting both felids.

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.

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.

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.

Plasmodium cynomolgi: immunization of a rhesus monkey with exoerythrocytic stages cultured in autologous hepatocytes.

To investigate the immune response to exoerythrocytic stages of malaria parasites, a rhesus monkey was immunized with autologous primary hepatocyte cultures infected with 7-day-old liver stage parasites of Plasmodium cynomolgi. A primary antibody response against EE stage antigens was obtained, and boosted after injection of homologous viable sporozoites. Antibodies directed against sporozoites and blood stages were also detected. The polyvalent immune response observed demonstrates the antigenicity of the liver stages and suggests their involvement in the general immune response against malaria.

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.

Ultrastructure of malaria-infected erythrocytes.

Knobs, caveolae, caveola-vesicle complexes, cytoplasmic clefts, and electron-dense material are five major ultrastructural changes found in the membrane skeleton and cytoplasm of erythrocytes infected with species of primate malaria. Knobs are electron-dense, conical evaginations of the erythrocyte surface, which are believed to mediate cytoadherence and sequestration of Plasmodium falciparum-infected erythrocytes. Caveolae and caveola-vesicle complexes are flask-shaped invaginations of the membrane skeleton, which may be involved in the uptake or export of host- or parasite-derived substances. Cytoplasmic clefts are flattened or circular membranous structures found in the erythrocyte cytoplasm between the intracellular parasite and the host cell surface. The clefts are variable in length and bounded by two or more membranes. Fine, granular electron-dense material is often found on the cytoplasmic face of clefts or in amorphous packets in the erythrocyte cytoplasm. Immunocytochemistry has demonstrated that all of these ultrastructural changes are associated with the trafficking and interaction of specific malarial antigens with the host erythrocyte.