Plasmodium relictum (lineage SGS1) and Plasmodium ashfordi (lineage GRW2): the effects of the co-infection on experimentally infected passerine birds

The effects of avian malaria parasites of the genus Plasmodium on their hosts are insufficiently understood. This is particularly true for malarial co-infections, which predominant in many bird populations. We investigated effects of primary co-infection of Plasmodium relictum (lineage SGS1) and Plasmodium ashfordi (GRW2) on experimentally infected naive juveniles of siskin Spinus spinus, crossbill Loxia curvirostra and starling Sturnus vulgaris. All siskins and crossbills were susceptible but starlings resistant to both these infections. A general pattern of the co-infections was that heavy parasitemia (over 35% during peaks) of both parasites developed in both susceptible host species. There were no significant effects of the co-infections on mean body mass of the majority of infected birds. Mean haematocrit value decreased approximately 1.5 and 3 times in siskins and crossbills at the peak of parasitemia, respectively. Mortality was recorded among infected crossbills. We conclude that co-infections of P. relictum and P. ashfordi are highly virulent and act synergetically during primary infections in some but not all passerine birds.     

Effect of repeated exposure to Plasmodium relictum (lineage SGS1) on infection dynamics in domestic canaries

Parasites are known to exert strong selection pressures on their hosts and, as such, favour the evolution of defence mechanisms. The negative impact of parasites on their host can have substantial consequences in terms of population persistence and the epidemiology of the infection. In natural populations, however, it is difficult to assess the cost of infection while controlling for other potentially confounding factors. For instance, individuals are repeatedly exposed to a variety of parasite strains, some of which can elicit immunological memory, further protecting the host from subsequent infections. Cost of infection is, therefore, expected to be particularly strong for primary infections and to decrease for individuals surviving the first infectious episode that are re-exposed to the pathogen. We tested this hypothesis experimentally using avian malaria parasites (Plasmodium relictum-lineage SGS1) and domestic canaries (Serinus canaria) as a model. Hosts were infected with a controlled dose of P. relictum as a primary infection and control birds were injected with non-infected blood. The changes in haematocrit and body mass were monitored during a 20 day period. A protein of the acute phase response (haptoglobin) was assessed as a marker of the inflammatory response mounted in response to the infection. Parasite intensity was also monitored. Surviving birds were then re-infected 37 days post primary infection. In agreement with the predictions, we found that primary infected birds paid a substantially higher cost in terms of infection-induced reduction in haematocrit compared with re-exposed birds. After the secondary infection, re-exposed hosts were also able to clear the infection at a faster rate than after the primary infection. These results have potential consequences for the epidemiology of avian malaria, since birds re-exposed to the pathogen can maintain parasitemia with low fitness costs, allowing the persistence of the pathogen within the host population.     

Plasmodium relictum infection and MHC diversity in the house sparrow (Passer domesticus)

Antagonistic coevolution between hosts and parasites has been proposed as a mechanism maintaining genetic diversity in both host and parasite populations. In particular, the high level of genetic diversity usually observed at the major histocompatibility complex (MHC) is generally thought to be maintained by parasite-driven selection. Among the possible ways through which parasites can maintain MHC diversity, diversifying selection has received relatively less attention. This hypothesis is based on the idea that parasites exert spatially variable selection pressures because of heterogeneity in parasite genetic structure, abundance or virulence. Variable selection pressures should select for different host allelic lineages resulting in population-specific associations between MHC alleles and risk of infection. In this study, we took advantage of a large survey of avian malaria in 13 populations of the house sparrow (Passer domesticus) to test this hypothesis. We found that (i) several MHC alleles were either associated with increased or decreased risk to be infected with Plasmodium relictum, (ii) the effects were population specific, and (iii) some alleles had antagonistic effects across populations. Overall, these results support the hypothesis that diversifying selection in space can maintain MHC variation and suggest a pattern of local adaptation where MHC alleles are selected at the local host population level.     

The avian malaria parasite Plasmodium gallinaceum causes marked structural changes on the surface of its host erythrocyte

Using a combination of atomic force, scanning and transmission electron microscopy, we found that avian erythrocytes infected with the avian malaria parasite Plasmodium gallinaceum develop 60 nm wide and 430 nm long furrow-like structures on the surface. Furrows begin to appear during the early trophozoite stage of the parasite’s development. They remain constant in size and density during the course of parasite maturation and are uniformly distributed in random orientations over the erythrocyte surface. In addition, the density of furrows is directly proportional to the number of parasites contained within the erythrocyte. These findings suggest that parasite-induced intraerythrocytic processes are involved in modifying the surface of host erythrocytes. These processes may be analogous to those of the human malaria parasite P. falciparum, which induces knob-like protrusions that mediate the pathogenic adherence of parasitized erythrocytes to microvessels. Although P. gallinaceum-infected erythrocytes do not seem to adhere to microvessels in the host chicken, the furrows might be involved in the pathogenesis of P. gallinaceum infections by some other mechanism involving host-pathogen interactions.     

Gnathostoma binucleatum: Pathological and parasitological aspects in experimentally infected dogs

Lesions and antibody kinetics produced by inoculation of Gnathostoma binucleatum larvae into dogs are described, as well as the morphology of the recovered parasites. In four out of five infected bitches parasite phases were found in the stomach. Only one bitch eliminated eggs and adult parasite phases in feces. In this bitch, the prepatency period lasted 22 weeks and the patency period 14 weeks. Necropsy results showed a copiously vascularized 8-cm diameter fibrous nodule lodged in the greater curvature of the stomach. Two bitches that eliminated no eggs showed 1- to 2-cm diameter nodules on the gastric wall, with five juvenile phases in each. One bitch that eliminated no eggs and exhibited no gastric nodules showed juvenile parasites on the gastric wall. Results confirm dogs as definitive hosts of this parasite. New data on the pathological and parasitological aspects of canine gnathostomosis are presented.     

Antibody response of naturally infected individuals to recombinant Plasmodium vivax apical membrane antigen-1

In the present study, we evaluate the naturally acquired antibody response to the Plasmodium vivax apical membrane antigen 1 (PvAMA-1), a leading vaccine candidate against malaria. The gene encoding the PvAMA-1 ectodomain region (amino acids 43-487) was cloned by PCR using genomic DNA from a Brazilian individual with patent P. vivax infection. The predicted amino acid sequence displayed a high degree of identity (97.3%) with a previously published sequence from the P. vivax Salvador strain. A recombinant protein representing the PvAMA-1 ectodomain was expressed in Escherichia coli and refolded. By ELISA, this recombinant protein reacted with 85 and 48.5% of the IgG or IgM antibodies, respectively, from Brazilian individuals with patent P. vivax malaria. IgG1 was the predominant subclass of IgG. The frequency of response increased according to the number of malaria episodes, reaching 100% in individuals in their fourth malaria episode. The high degree of recognition of PvAMA-1 by human antibodies was confirmed using a second recombinant protein expressed in Pichia pastoris (PV66/AMA-1). The observation that recognition of the bacterial recombinant PvAMA-1 was only slightly lower than that of the highly immunogenic 19kDa C-terminal domain of the P. vivax Merozoite Surface Protein-1 was also important. DNA sequencing of the PvAMA-1 variable domain from 20 Brazilian isolates confirmed the limited polymorphism of PvAMA-1 suggested by serological analysis. In conclusion, we provide evidence that PvAMA-1 is highly immunogenic during natural infection in humans and displays limited polymorphism in Brazil. Based on these observations, we conclude that PvAMA-1 merits further immunological studies as a vaccine candidate against P. vivax malaria.     

The role of ICAM-1 in Plasmodium falciparum cytoadherence

Parasite sequestration at microvascular sites is a fundamental phenomenon in the manifestation of the symptoms of malaria and the progression to severe disease. Here, we review the endothelial cell-expressed intercellular adhesion molecule-1 (ICAM-1) and its role in mediating the interaction between the parasitised red blood cell (PRBC) and the vascular endothelium. We highlight the nature of the interaction between ICAM-1 and the parasite-expressed PfEMP-1 molecule at the molecular level. The review also discusses the complexity of the PRBC-endothelial cell interaction and the mechanisms that underlie parasite cytoadherence.     

Specific erythrocyte binding capacity and biological activity of Plasmodium falciparum erythrocyte binding ligand 1 (EBL-1)-derived peptides

Erythrocyte binding ligand 1 (EBL-1) is a member of the ebl multigene family involved in Plasmodium falciparum invasion of erythrocytes. We found that five EBL-1 high-activity binding peptides (HABPs) bound specifically to erythrocytes: 29895 ((41)HKKKSGELNNNKSGILRSTY(60)), 29903 ((201)LYECGK-KIKEMKWICTDNQF(220)), 29923 ((601)CNAILGSYADIGDIVRGLDV(620)), 29924((621)WRDINTNKLSEK-FQKIFMGGY(640)), and 30018 ((2481)LEDIINLSKKKKKSINDTSFY(2500)). We also show that binding was saturable, not sialic acid-dependent, and that all peptides specifically bound to a 36-kDa protein on the erythrocyte membrane. The five HABPs inhibited in vitro merozoite invasion depending on the peptide concentration used, suggesting their possible role in the invasion process.     

Social interactions modulate the virulence of avian malaria infection

There is an increasing understanding of the context-dependent nature of parasite virulence. Variation in parasite virulence can occur when infected individuals compete with conspecifics that vary in infection status; virulence may be higher when competing with uninfected competitors. In vertebrates with social hierarchies, we propose that these competition-mediated costs of infection may also vary with social status. Dominant individuals have greater competitive ability than competing subordinates, and consequently may pay a lower prevalence-mediated cost of infection. In this study we investigated whether costs of malarial infection were affected by the occurrence of the parasite in competitors and social status in domestic canaries (Serinus canaria). We predicted that infected subordinates competing with non-infected dominants would pay higher costs than infected subordinates competing with infected dominants. We also predicted that these occurrence-mediated costs of infection would be ameliorated in infected dominant birds. We found that social status and the occurrence of parasites in competitors significantly interacted to change haematocrit in infected birds. Namely, subordinate and dominant infected birds differed in haematocrit depending on the infection status of their competitors. However, in contrast to our prediction, dominants fared better with infected subordinates, whereas subordinates fared better with uninfected dominants. Moreover, we found additional effects of parasite occurrence on mortality in canaries. Ultimately, we provide evidence for costs of parasitism mediated by social rank and the occurrence of parasites in competitors in a vertebrate species. This has important implications for our understanding of the evolutionary processes that shape parasite virulence and group living.     

Food availability and competition do not modulate the costs of Plasmodium infection in dominant male canaries

Understanding the different factors that may influence parasite virulence is of fundamental interest to ecologists and evolutionary biologists. It has recently been demonstrated that parasite virulence may occur partly through manipulation of host competitive ability. Differences in competitive ability associated with the social status (dominant or subordinate) of a host may determine the extent of this competition-mediated parasite virulence. We proposed that differences between subordinate and dominant birds in the physiological costs of infection may change depending on the level of competition in social groups. We observed flocks of domestic canaries to determine dominant or subordinate birds, and modified competition by providing restricted (high competition) or ad libitum food (low competition). Entire flocks were then infected with either the avian malaria parasite, Plasmodium relictum or a control. Contrary to our predictions we found that the level of competition had no effect on the outcome of infection for dominant or subordinate birds. We found that dominant birds appeared to suffer greater infection mediated morbidity in both dietary treatments, with a higher and more sustained reduction in haematocrit, and higher parasitaemia, than subordinates. Our results show that dominance status in birds can certainly alter parasite virulence, though the links between food availability, competition, nutrition and virulence are likely to be complex and multifaceted.     

Trials for the co-expression of the merozoite surface protein-1 and circumsporozoite protein genes of Plasmodium vivax

Merozoite surface protein-1 (MSP-1), a major asexual blood stage antigen, and circumsporozoite protein (CSP), a component of sporozoites that includes a Plasmodium vivax B-cell epitope, are strong candidates for use in a malaria vaccine. A chimeric recombinant gene containing portions of both msp-1 and csp from P. vivax separated by Pro-Gly linker motif was generated. The construct gene was named mlc (msp-1, linker, and csp). The MLC chimeric recombinant protein had a molecular weight of approximately 25 kDa when expressed in Escherichia coli, as determined with sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) analysis. The purified chimeric protein reacted with the sera of patients infected with P. vivax but not with the sera of uninfected patients according to western blot analysis. The chimeric protein reacted well with sera of malaria patients (109/115, 94.78%) as assessed with enzyme-linked immunosorbent assay (ELISA). BALB/c mice that were orally immunized with the MLC chimeric recombinant protein successfully produced antigen-specific antibodies. Additionally, levels of the Th1-associated cytokines IL-12(p40), TNF-α, and IFN-γ were significantly increased in the spleens of the BALB/c mice. Therefore, the E. coli-expressed MLC chimeric recombinant protein might be used as a valuable vaccine candidate for oral immunization against vivax malaria.     

Plasmodium vivax: genetic diversity of the apical membrane antigen-1 (AMA-1) in isolates from India

Malaria parasites exhibit sequence diversity for a number of stage specific antigens. Several studies have proved that apical membrane antigen-1 (AMA-1) is an effective target for eliciting a protective immune response in humans and other experimental animals. We have investigated the sequence variation in Plasmodium vivax AMA-1 (Pv AMA-1) from different Indian isolates. This is the first study of its kind for the nearly full length Pv AMA-1 from India. Our analysis reveals greater degree of genetic diversity in Pv AMA-1 than reported so far and identifies five novel haplotypes. This is significant to establish the antigenic repertoire of isolates in a malaria endemic country like India.     

Plasmodium falciparum: differing effects of non-esterified fatty acids and phospholipids on intraerythrocytic growth in serum-free medium

Different combinations of non-esterified fatty acids (NEFA) had variable effects on intraerythrocytic growth of Plasmodium falciparum. All stages of the parasite cultured in medium supplemented with cis-9-octadecenoic acid (C18:1-cis-9), hexadecanoic acid (C16:0), phospholipids (Pld) and bovine albumin free of NEFA were similar to those grown in complete growth medium. Three typical growth patterns indicating suppressed schizogony (SS), suppressed formation of merozoites (SMF), and inhibited invasion of merozoites (IMI) resulted from culture in other combinations of lipids. Unsaturated or saturated NEFA with longer or shorter carbon chains than C18:1-cis-9 or C16:0, higher degree of unsaturation, and trans-forms mainly resulted in SS and SMF effects. However, IMI or partial IMI was observed with tetradecanoic acid or octadecanoic acid enriched with C18:1-cis-9, and cis-9-hexadecenoic acid plus C16:0. Isoforms of C18:1-cis-9 also mainly resulted in partial IMI. SMF also occurred with C18:1-cis-9 plus C16:0 in the absence of Pld. Thus different NEFA exerted distinct roles in erythrocytic growth of the parasite by sustaining development at different stages.     

Crystal structure of a Fab complex formed with PfMSP1-19, the C-terminal fragment of merozoite surface protein 1 from Plasmodium falciparum: a malaria vaccine candidate

Merozoite surface protein 1 (MSP1) is the major protein component on the surface of the merozoite, the erythrocyte-invasive form of the malaria parasite Plasmodium. Present in all species of Plasmodium, it undergoes two distinct proteolytic maturation steps during the course of merozoite development that are essential for invasion of the erythrocyte. Antibodies specific for the C-terminal maturation product, MSP1-19, can inhibit erythrocyte invasion and parasite growth. This polypeptide is therefore considered to be one of the more promising malaria vaccine candidates. We describe here the crystal structure of recombinant MSP1-19 from P.falciparum (PfMSP1-19), the most virulent species of the parasite in humans, as a complex with the Fab fragment of the monoclonal antibody G17.12. This antibody recognises a discontinuous epitope comprising 13 residues on the first epidermal growth factor (EGF)-like domain of PfMSP1-19. Although G17.12 was raised against the recombinant antigen expressed in an insect cell/baculovirus system, it binds uniformly to the surface of merozoites from the late schizont stage, showing that the cognate epitope is exposed on the naturally occurring MSP1 polypeptide complex. Although the epitope includes residues that have been mapped to regions recognised by invasion-inhibiting antibodies studied by other workers, G17.12 does not inhibit erythrocyte invasion or MSP1 processing.     

Genetic diversity in merozoite surface protein (MSP)-1 and MSP-2 genes of Plasmodium falciparum in a major endemic region of Iran

Merozoite surface protein-1 (MSP-1) and merozoite surface protein-2 (MSP-2) were used to develop vaccines and to investigate the genetic diversity in Plasmodium falciparum malaria in Iran. Nested polymerase chain reaction amplification was used to determine polymorphisms of block 2 of the MSP-1 and the central domain of MSP-2 genes. A total of 67 microscopically positive P. falciparum infected individuals from a major endemic region, southeast Iran, were included in this trial. Nine alleles of MSP-1 and 11 alleles of MSP-2 were identified. The results showed that amplified product from these surface antigen genes varied in size and there was specific pattern for each isolate. Besides, regarding this pattern, 23 multiple infections with at least 2 alleles were observed. While the endemic regions of malaria in Iran is classified in low to moderate group, but extensive polymorphism was observed for each marker and the MSP-2 central repeat was the most diverse that could be considered in designing malaria vaccine.     

Detection of Candida species by polymerase chain reaction (PCR) in blood samples of experimentally infected mice and patients with suspected candidemia

In this study, we have established and evaluated a genus-specific polymerase chain reaction (PCR) and species-specific nested PCRs for the detection of Candida species in blood samples of neutropenic mice and patients suspected of candidemia. DNA segments of the gene encoding cytochrome P450 L1A1 were targeted for amplification by using genus and species-specific primers. As compared to the genus-specific PCR, the species-specific nested PCRs improved the sensitivity by 10 times with the detection limit < 10 yeast cells. Of the 18 blood samples tested daily over a period of 8 days following Candida albicans infection in neutropenic mice, four samples were positive by genus-specific PCR and 11 were positive by species-specific nested PCR. The PCR results were correlated with culture findings obtained on blood samples. Two of the three blood culture-positive samples were positive by genus-specific PCR and all the three with species-specific nested PCR. Among 15 mice, which were negative by blood culture but had C. albicans isolated from visceral organs, 2 and 8 mice yielded positive results by genus-specific PCR and species-specific nested PCR, respectively. Consistent with the results of the animal study, species-specific nested PCR yielded much higher positivity as compared to culture (52.2% versus 21.2%) in patients suspected for candidemia. Moreover, 8 specimens which were negative for Candida by genus-specific PCR became positive by species-specific nested PCR. No correlation was apparent between PCR positivity and Candida antigen titers. The results suggest that nested PCR is a sensitive technique for the detection of Candida species from blood samples, and thus it may have application in the diagnosis of suspected cases of candidemia and candidiasis.     

Molecular phylogeny of Phebalium (Rutaceae: Boronieae) and related genera based on the nrDNA regions ITS 1+2

Parsimony analyses of the internal transcribed spacer regions of nuclear ribosomal DNA (ITS 1 & ITS 2) for 38 taxa sampled from the Phebalium group (Rutaceae: Boronieae) and two outgroups confirm that, with the exception of Phebalium sensu stricto and Rhadinothamnus, six of the currently recognised genera within the group are monophyletic. The data indicate that Phebaliums. str. is paraphyletic with respect to Microcybe, and Rhadinothamnus is paraphyletic with respect to Chorilaena. Rhadinothamnus and Chorilaena together are the sister group to Nematolepis. Drummondita, included as an outgroup taxon, clustered within the ingroup as sister to Muiriantha and related to Asterolasia.The phylogeny suggests that the evolution of major clades within a number of these genera (e.g. Phebalium) relates to vicariance events between eastern and south-western Australia. Leionema is an eastern genus, with the most basal taxon being the morphologically distinct Leionema ellipticum from northern Queensland. Leionema also includes one species from New Zealand, but this species (as with some others) proved difficult to sequence and its phylogenetic position remains unknown. Taxonomic changes at the generic level are recommended.The authors wish to thank Paul G.Wilson, PERTH, for advice and discussion, and Paul Forster, BRI, for collecting and providing material of Leionema ellipticum. The project was supported by a Melbourne University Postgraduate Award (to BM), the Australian Biological Resources Study (ABRS), Australian Systematic Botany Society and Wolf Den (Australia) Investments.     

Tolerance of Bt corn (MON 810) to maize stem borer, Chilo partellus (Lepidoptera: Pyralidae)

Transgenic corn (MON 810), expressing the Bacillus thuringiensis (Bt) protein, Cry1Ab, was evaluated under greenhouse conditions for its tolerance to the maize stem borer, Chilo partellus. Bt corn (MON 810) provided effective protection against the stem borer even under a high level of larval infestation in the greenhouse. The observed tolerance is examined and discussed in the light of the susceptibility of C. partellus to the Cry1Ab protein in laboratory bioassays. The implications of the tissue concentrations of Cry1Ab in MON 810, and baseline susceptibility recorded in the current study, for insect-resistance management are discussed.