Truncation of Plasmodium berghei merozoite surface protein 8 does not affect in vivo blood-stage development

Merozoite surface protein 8 (MSP8) has shown promise as a vaccine candidate in the Plasmodium yoelii rodent malaria model and has a proposed role in merozoite invasion of erythrocytes. However, the temporal expression and localisation of MSP8 are unusual for a merozoite antigen. Moreover, in Plasmodium falciparum the MSP8 gene could be disrupted with no apparent effect on in vitro growth. To address the in vivo function of full-length MSP8, we truncated MSP8 in the rodent parasite Plasmodium berghei. PbΔMSP8 disruptant parasites displayed a normal blood-stage growth rate but no increase in reticulocyte preference, a phenomenon observed in P. yoelii MSP8 vaccinated mice. Expression levels of erythrocyte surface antigens were similar in P. berghei wild-type and PbΔMSP8-infected erythrocytes, suggesting that a parasitophorous vacuole function for MSP8 does not involve global trafficking of such antigens. These data demonstrate that a full-length membrane-associated form of PbMSP8 is not essential for blood-stage growth.

Undiagnosed and potentially lethal parasite infections among immigrants and refugees in Australia

Background: Intestinal parasite infections are a major cause of ill health in many resource-poor countries. This study compares the types and rates of these infections and their risk factors in recently arrived and long-term immigrants in Australia.

Method: Cross-sectional surveys of 127 East African and 234 Cambodian immigrants and refugees were undertaken in 2000 and 2002, respectively, to assess the burden of intestinal parasites and collect demographic information. Serum samples were assessed for eosinophilia and Strongyloides stercoralis and Schistosoma antibodies, and feces examined for ova, cysts, and parasites.

Results: Intestinal parasites were identified in 77/117 fecal samples from East African and in 25/204 samples collected from Cambodian participants. Eleven percent (14/124) of East Africans and 42% (97/230) of Cambodians had positive or equivocal serology for S stercoralis. Schistosoma serology was positive or equivocal in 15% (19/124) of East African participants.

Conclusion: Potentially serious intestinal parasite infections are common among recent and longer term immigrants despite multiple visits to health care providers. Immigrants and refugees from high-risk countries would benefit from comprehensive health checks soon after resettlement.

Spatial ecology of a root parasite : from pattern to process

Occurrence patterns of parasitic plants are constrained by the distribution of suitable hosts and movement patterns of seed vectors and, accordingly, represent a simplified system to study many aspects of spatial ecology and determinants of distribution. Previous work has focused on the aerially hemiparasitic mistletoes, and it is unclear whether root parasites are affected by similar factors. Here, we evaluate spatial patterns in the root parasitic Santalum lanceolatum in an arid shrubland in north-western New South Wales, central Australia. In this region, the principal host is a long-lived nitrogen fixing shrub Acacia tetragonophylla closely associated with ephemeral creek-lines. The location of 765 individuals of both species was mapped along a 250-m section of creek-line using a total survey station, and occurrence patterns of the root parasite related to host distribution and landscape context. We used Ripley's K-function and the O-ring statistic to determine whether the distribution of S. lanceolatum was random, aggregated or regular; the spatial scales at which these patterns occurred; and to quantify any spatial associations between the parasite and its host, A. tetragonophylla. While acacias were closely associated with the creek-line, S. lanceolatum plants were more tightly clustered, displaying significant clustering at two spatial scales (1.2 m and 8.8 m). We suggest that host quality may act as an important constraint, with only those acacias growing in or near the creek-line being physiologically capable of supporting a parasite to maturity. Insights gained from spatial analysis are used to guide ongoing research in this system, and highlight the utility of the O-ring statistic for understanding patterns of distribution affected by multiple processes operating at critical scales.

Environmental variation and the maintenance of polymorphism : the effect of ambient light spectrum on mating behaviour and sexual selection in guppies

The intensity of sexual selection is influenced by environmental conditions because these conditions influence signal propagation and the risks of the signal being exploited by predators and parasites. We explore the possibility that spatial or temporal heterogeneity in environmental signalling conditions (in this case light spectrum) may induce fluctuating sexual selection on male behaviour and ornamentation in guppies. We used shade cloth and filters to experimentally manipulate light spectrum, mimicking conditions found naturally: early morning/late afternoon light (SC treatment), midday forest shade (F89 filter treatment) and midday woodland shade (F55 filter treatment). Females were more responsive to male courtship and males were less likely to attempt sneak copulations under F55 light than the other two treatments. By contrast, male display rate was not influenced by treatment. Females tended to prefer the same males under SC and F55 light, but attractiveness in these treatments was unrelated to attractiveness under F89 light. There were similarities among treatments in the traits that females preferred: females preferred males with larger areas of orange in all three treatments. There were, however, also some differences, including preference for larger males under F89 light and for smaller males under the other treatments. Overall, the influence of ambient light spectrum on the relative importance of mate choice and male sneak copulation may have important implications for the mode and strength of sexual selection in different environments. The findings on attractiveness and preference functions, however, suggest that light spectrum only weakly affects the direction of sexual selection by female choice.

Control of ovarian development in the yabby (Cherax destructor)

A study under controlled conditions of ovarian development and rematuration in the yabby (Cherax destructot) was undertaken. The purpose of the study was to improve fundamental understanding of the reproductive biology of the species and provide a basis for application to hatchery management in culture. A review was made of the current status of yabby culture in Australia and the present understanding of reproductive biology of decapod Crustacea. The review emphasised factors controlling several aspects of ovarian development, in particular the processes of vitellogenesis. The subsequent study was designed within the context of current hatchery practice and was based on existing knowledge of decapod reproduction, The sexual differentiation of the yabby after hatching was investigated by serial histological sections, and experiments were carried out to investigate the possibility of sex reversal of males. Most of this Investigation was concerned with removing the influence of the androgenic gland in directing male development, with the intent of observing the development of the elementary gonadal tissue into ovary. It was found that in contrast to other crustacean species, the sex of the yabby becomes fixed before the development of external secondary sexual characteristics, and before the androgenic gland can be discerned. Ovarian tissue developed in females at less than 8 weeks after hatching. A preliminary examination was undertaken for feminising parasites in gonadal tissue of a hermaphrodite yabby. Investigation of the ovary after spawning demonstrated that whilst the female was held under constant conditions of temperature and photoperiod, little rematuration occurred. Except for generation of previtellogenic oocytes during the first two days, the gonaciosomatic index remained low for up to 5 months after spawning. If the temperature of the female was reduced to 10°C and maintained constant, the previtellogenic oocytes were partially resorbed over a three week period. Rematuration then commenced, albeit at a low rate because of the reduced temperature, A method for standardising gonadosomatic indices was developed which took into account differences in hepatopancreatic nutrient reserves of individuals and loss of one or more appendages. This part of the study also considered constraints to rematuration and developed a method of accounting for differences in the ability of females to remature after spawning. Experiments were carried out to investigate the effect of crowding and temperature manipulation on initiating ovarian rematuration and to determine the rate of rematuration at 22°C once initiated. The duration of low temperature had no effect on rematuration; an overnight cooling was sufficient to initiate the process, Rematuration to the end of stage 2 vltellogenesis was substantially complete within 10 days. Crowding of females suppressed rematuration, but less than ideal water quality was not found to have any effect. The presence of a male initiated rematuration at a similar rate, but also led to stage 3 vitetlogenesis and spawning. A study was made of the pheromonal influence of the male through water borne factors without success. Rematuration could not be induced in ovigerous females. The literature review indicated that ovarian rematuration was under the control of an ovary stimulating hormone produced by the thoracic nerve ganglia. Attempts were therefore made to stimulate ovarian rematuration by incorporating the thoracic nerve into the diet of females. Attempts were also made to induce the release of ovary stimulating hormone from the thoracic nerve with 5-hydroxytryptamine, and also with octopamine. No effects were found, but a significant difference between the neurophysiology of the yabby and northern hemisphere crayfish was observed, and the implications of this finding are discussed. The study did not produce any conclusive evidence of an ovary stimulating hormone for the yabby. A model of ovarian rematuration which collects the findings of the experimental investigations was developed, and was used to suggest a hatchery broodstock management protocol. This model differs from existing models in that rematuration triggers and nutritional status are considered.

Weaning and growth of Anguilla australis glass eels and elvers

Anguilla australis glass eels proved to be resilient and present strong aquaculture potential. General husbandry techniques, anaesthesia and prophylactic treatments were established for glass eels between 0.1 g and 2.0 g and elvers between 2.0 g and 8.0 g, caught in rivers and estuaries along the South East Coast of Victoria. The protozoan parasites Ichthyobodo and Trichodina were found to be present on arrival to the hatchery developed during different rearing treatments, and were successfully eradicated. A. australis glass eels accepted artificial food, but it was recommended first be fed a preweaning diet of minced fish flesh. A weaning regime from minced fish flesh to commercially available eel grower mash, over 15 days was established. Growth rate proved to be highly variable, both between and within groups. The highest growth rate of 2.71%/day was found when the natural diet of minced fish and Artemia was fed. The maximum growth rate when reared on an artificial diet of 1.63%/day was observed at 25°C. Growth was affected by the presence or absence of a preweaning diet, weaning diet, weaning period, temperature, but not by size or density. Once weaned, glass eels were found to perform better on commercially available grower mash than on the minced fish flesh, which was used to aid in weaning them to artificial diets. Of the water quality parameters measured stocking density was found to affect pH, Total Ammonia Nitrogen, Total Phosphorus, and Dissolved Oxygen, through not to an extent which affected growth.

Other communities : Box mistletoe (Amyema miquelii) parasitism is not detrimental to the health of grey box (Eucalyptus microcarpa) trees at a regional scale

Mistletoes are hemiparasites that occur worldwide in many types of forest, woodland and shrubland ecosystems (Watson 2001). Some species are regarded as pests due to their detrimental effects on host species (Hawksworth 1983; Reid & Yan 2000). Heavy infestations can affect the growth, productivity and form of host trees, and may cause host death (Reid et al. 1994; Shaw et al.2004, 2008). In south-eastern Australia, mistletoes often are visibly obvious in trees along roadsides, in paddocks and on the margins of open forests; and concerns have been expressed about their potentially detrimental effects on host trees.Despite this, little quantitative information is available on the effects of mistletoes on tree health and mortality (Reid et al. 1994). Are detrimental effects widespread or localized? A first step is to assess whether trees parasitized by mistletoe are less healthy than those without such parasites. Here, we investigate the relationship between parasitism by Box Mistletoe (Amyema miquelii (Lehm. ex Miq.) Tiegh.), a common species in south-eastern Australia, and the health of trees of a widespread host species, Grey Box (Eucalyptus microcarpa (Maiden) Maiden), across a large geographic region.

Plasmodium falciparum induces reorganization of host membrane proteins during intraerythrocytic growth

The virulence of the malaria parasite, Plasmodium falciparum, is due in large part to the way in which it modifies the membrane of its erythrocyte host. In this work we have used confocal microscopy and fluorescence recovery after photo-bleaching to examine the lateral mobility of host membrane proteins in erythrocytes infected with P falciparum at different stages of parasite growth. The erythrocyte membrane proteins band 3 and glycophorin show a marked decrease in mobility during the trophozoite stage of growth. Erythrocytes infected with a parasite strain that does not express the knob-associated histidine-rich protein show similar effects, indicating that this parasite protein does not contribute to the immobilization of the host proteins. Erythrocytes infected with ring-stage parasites exhibit intermediate mobility indicating that the parasite is able to modify its host prior to its active feeding stage.

A conserved region in the EBL proteins Is implicated in microneme targeting of the malaria parasite Plasmodium falciparum

The proliferation of the malaria parasite Plasmodium falciparum within the human host is dependent upon invasion of erythrocytes. This process is accomplished by the merozoite, a highly specialized form of the parasite. Secretory organelles including micronemes and rhoptries play a pivotal role in the invasion process by storing and releasing parasite proteins. The mechanism of protein sorting to these compartments is unclear. Using a transgenic approach we show that trafficking of the most abundant micronemal proteins (members of the EBL-family: EBA-175, EBA-140/BAEBL, and EBA-181/JSEBL) is independent of their cytoplasmic and transmembrane domains, respectively. To identify the minimal sequence requirements for microneme trafficking, we generated parasites expressing EBAGFP chimeric proteins and analyzed their distribution within the infected erythrocyte. This revealed that: (i) a conserved cysteine-rich region in the ectodomain is necessary for protein trafficking to the micronemes and (ii) correct sorting is dependent on accurate timing of expression.

Recent advances of metal binding protein lactoferrin as an anti-microbial agent

Lactoferrin (Lf) is present in milk and gland secretions and serve as an antimicrobial function. Insufficient amounts of Lf in some secretions also appear to correlate with certain health problems. Protection against gastroenteritis is the most likely biologically relevant activity of lactoferrin. Multiple in vitro and animal studies have shown a protective effect of lactoferrin on infections with enteric microorganisms, including rotavirus, Giardia, Shigella, Salmonella and the diarrheagenic Escherichia coli. Lactoferrin has two major effects on enteric pathogens: it inhibits growth and it impairs function of surface expressed virulence factors thereby decreasing their ability to adhere or to invade mammalian cells. Lf also inhibits several species of fungi and certain parasites. This review covers the role of Lf in clearing the parasitic infections. The mechanism by which lactoferrin inhibits some parasites may be via stimulation of the process of phagocytosis, whereby immune cells engulf and digest foreign organisms. Trichomonas vaginalis is a protozoan responsible for the number one, non-viral sexually transmitted disease. In this review, we also discussed the role of Lf in cervical infections.

The clp chaperones and proteases of the human malaria parasite Plasmodium falciparum

The Clp chaperones and proteases play an important role in protein homeostasis in the cell. They are highly conserved across prokaryotes and found also in the mitochondria of eukaryotes and the chloroplasts of plants. They function mainly in the disaggregation, unfolding and degradation of native as well as misfolded proteins. Here, we provide a comprehensive analysis of the Clp chaperones and proteases in the human malaria parasite Plasmodium falciparum. The parasite contains four Clp ATPases, which we term PfClpB1, PfClpB2, PfClpC and PfClpM. One PfClpP, the proteolytic subunit, and one PfClpR, which is an inactive version of the protease, were also identified. Expression of all Clp chaperones and proteases was confirmed in blood-stage parasites. The proteins were localized to the apicoplast, a non-photosynthetic organelle that accommodates several important metabolic pathways in P. falciparum, with the exception of PfClpB2 (also known as Hsp101), which was found in the parasitophorous vacuole. Both PfClpP and PfClpR form mostly homoheptameric rings as observed by size-exclusion chromatography, analytical ultracentrifugation and electron microscopy. The X-ray structure of PfClpP showed the protein as a compacted tetradecamer similar to that observed for Streptococcus pneumoniae and Mycobacterium tuberculosis ClpPs. Our data suggest the presence of a ClpCRP complex in the apicoplast of P. falciparum.

Functional analysis of the leading malaria vaccine candidate AMA-1 reveals an essential role for the cytoplasmic domain in the invasion process

A key process in the lifecycle of the malaria parasite Plasmodium falciparum is the fast invasion of human erythrocytes. Entry into the host cell requires the apical membrane antigen 1 (AMA-1), a type I transmembrane protein located in the micronemes of the merozoite. Although AMA-1 is evolving into the leading blood-stage malaria vaccine candidate, its precise role in invasion is still unclear. We investigate AMA-1 function using live video microscopy in the absence and presence of an AMA-1 inhibitory peptide. This data reveals a crucial function of AMA-1 during the primary contact period upstream of the entry process at around the time of moving junction formation. We generate a Plasmodium falciparum cell line that expresses a functional GFP-tagged AMA-1. This allows the visualization of the dynamics of AMA-1 in live parasites. We functionally validate the ectopically expressed AMA-1 by establishing a complementation assay based on strain-specific inhibition. This method provides the basis for the functional analysis of essential genes that are refractory to any genetic manipulation. Using the complementation assay, we show that the cytoplasmic domain of AMA-1 is not required for correct trafficking and surface translocation but is essential for AMA-1 function. Although this function can be mimicked by the highly conserved cytoplasmic domains of P. vivax and P. berghei, the exchange with the heterologous domain of the microneme protein EBA-175 or the rhoptry protein Rh2b leads to a loss of function. We identify several residues in the cytoplasmic tail that are essential for AMA-1 function. We validate this data using additional transgenic parasite lines expressing AMA-1 mutants with TY1 epitopes. We show that the cytoplasmic domain of AMA-1 is phosphorylated. Mutational analysis suggests an important role for the phosphorylation in the invasion process, which might translate into novel therapeutic strategies.

RAP1 controls rhoptry targeting of RAP2 in the malaria parasite Plasmodium falciparum

Rhoptry associated protein 1 (RAP1) and 2 (RAP2), together with a poorly described third protein RAP3, form the low molecular weight complex within the rhoptries of Plasmodium falciparum. These proteins are thought to play a role in erythrocyte invasion by the extracellular merozoite and are important vaccine candidates. We used gene-targeting technology in P.falciparum blood-stage parasites to disrupt the RAP1 gene, producing parasites that express severely truncated forms of RAP1. Immunoprecipitation experiments suggest that truncated RAP1 species did not complex with RAP2 and RAP3. Consistent with this were the distinct subcellular localizations of RAP1 and 2 in disrupted RAP1 parasites, where RAP2 does not traffic to the rhoptries but is instead located in a compartment that appears related to the lumen of the endoplasmic reticulum. These results suggest that RAP1 is required to localize RAP2 to the rhoptries, supporting the hypothesis that rhoptry biogenesis is dependent in part on the secretory pathway in the parasite. The observation that apparently host-protective merozoite antigens are not essential for efficient erythrocyte invasion has important implications for vaccine design.

Biosynthesis, localization, and macromolecular arrangement of the plasmodium falciparum translocon of exported proteins (PTEX)

To survive within its host erythrocyte, Plasmodium falciparum must export hundreds of proteins across both its parasite plasma membrane and surrounding parasitophorous vacuole membrane, most of which are likely to use a protein complex known as PTEX (Plasmodium translocon of exported proteins). PTEX is a putative protein trafficking machinery responsible for the export of hundreds of proteins across the parasitophorous vacuole membrane and into the human host cell. Five proteins are known to comprise the PTEX complex, and in this study, three of the major stoichiometric components are investigated including HSP101 (a AAA+ ATPase), a protein of no known function termed PTEX150, and the apparent membrane component EXP2. We show that these proteins are synthesized in the preceding schizont stage (PTEX150 and HSP101) or even earlier in the life cycle (EXP2), and before invasion these components reside within the dense granules of invasive merozoites. From these apical organelles, the protein complex is released into the host cell where it resides with little turnover in the parasitophorous vacuole membrane for most of the remainder of the following cell cycle. At this membrane, PTEX is arranged in a stable macromolecular complex of >1230 kDa that includes an ∼600-kDa apparently homo-oligomeric complex of EXP2 that can be separated from the remainder of the PTEX complex using non-ionic detergents. Two different biochemical methods undertaken here suggest that PTEX components associate as EXP2-PTEX150-HSP101, with EXP2 associating with the vacuolar membrane. Collectively, these data support the hypothesis that EXP2 oligomerizes and potentially forms the putative membrane-spanning pore to which the remainder of the PTEX complex is attached.