Babesia bovis: effects of cysteine protease inhibitors on in vitro growth

In the present study, we examined the effects of four kinds of cysteine protease inhibitors (E64, E64d, leupeptin, and ALLN) on the in vitro asexual growth of Babesia bovis. Of these, only the lipophilic inhibitors, E64d and ALLN, were found to effectively inhibit the growth of B. bovis. In further experiments, E64d, but not ALLN, significantly suppressed the parasite’s invasion of host erythrocytes, while both chemicals, especially ALLN, inhibited the parasite’s replication within the infected erythrocytes. These data suggested the presence of cysteine protease(s) derived from B. bovis, in which the protease(s) would play important roles in the erythrocyte invasion and/or replication processes of the parasite.     

Effects of salinity on larval stages of the rhizocephalan barnacle Loxothylacus texanus: survival and metamorphosis in response to the host, Callinectes sapidus

It is known that the rhizocephalan barnacle Loxothylacus texanus infects the greater blue crab, Callinectes sapidus, in the Gulf of Mexico and adjacent waters, however, factors that affect the prevalence and distribution of this parasite, particularly the dispersive larval stages of this organism, are not well understood. In the current study, the effects of salinity on larval survival and the metamorphosis of L. texanus in response to postmolt host exoskeleton were examined. Acute and acclimated responses were similar. Larval survival was highest in the 20-35‰ range, with 100% mortality of nauplii at all salinities <20‰ and >50‰. L. texanus cyprids were able to metamorphose over a broad range of salinities (15-60‰). In several cases, metamorphosis was actually greatest at high salinities (40-50‰). These data predict that L. texanus larvae would be concentrated in portions of Gulf of Mexico waters with salinities >20‰ such as the mouths of estuaries and bays. Conversely, upper regions of estuaries may be inhospitable to the dispersive (naupliar) stage of the parasite and may serve as a refuge from infection for host crabs.     

Impacts of an endoparasitic copepod, Ismaila belciki, on the reproduction,growth and survivorship of its nudibranch host, Janolus fuscus

Copepods from the genus Ismaila are large endoparasites that inhabit the main body cavity and/or cerata of opisthobranch molluscs. These parasites exhibit many life history characteristics typically found in parasitic castrators, yet the actual impact of infection on reproduction, growth or survivorship of the hosts are unknown. On the Oregon (USA) coast, Ismaila belciki can infect over 80% of their hermaphroditic hosts, Janolus fuscus. In laboratory mating experiments, we compared the reproductive output (egg mass weight, number of egg capsules, number of viable embryos) and the gonadal somatic index of infected versus uninfected J. fuscus. Infected J. fuscus could produce viable sperm and copulate. Mating with an infected individual did not limit a sea slug’s reproductive output. However, infected J. fuscus had significantly lower reproductive output (by 34–54%), producing smaller egg masses with fewer capsules and viable embryos. Infected hosts had significantly lower gonadal somatic index than their uninfected counterparts, although there was no significant difference in gonadal somatic index between hosts with single and double infections. By collecting the egg sacs produced by the copepod parasite during experiments, we estimated that 25–34% of the host’s reproductive output is usurped by the parasite and re-directed to the parasite’s own reproduction. In the laboratory, infection did not alter growth in J. fuscus. However, infection significantly decreased survivorship in mature (but not immature) nudibranch hosts. These results suggest that I. belciki is not a true castrator, but it does reduce the reproductive output of its host and may therefore limit the natural population size of J. fuscus.     

Osmoregulatory disturbances induced by the parasitic barnacle Loxothylacus texanus (Rhizocephala) in the crab Callinectes rathbunae (Portunidae)

The osmoregulatory response of the blue crab Callinectes rathbunae parasitized with the rhizocephalan barnacle Loxothylacus texanus, and subjected to sudden salinity changes, was experimentally measured in the laboratory. Parasitized and control crabs were exposed to salinity changes every 3 h and their hemolymph osmolality measured. Two experiments, one with increasing salinity conditions (5‰, 12‰, 19‰, 25‰) and a second one with decreasing salinities (35‰, 25‰, 15‰, 5‰) were conducted. The results show that L. texanus significantly alters the hemolymph osmolality of C. rathbunae maintaining it at lower than normal levels. In the increasing salinity trial, the hypoosmotic hemolymph condition of parasitized crabs was present at all salinities tested, whereas in the decreasing salinity trial a significant effect was found only at salinities of 5‰ and 15‰. Since C. rathbunae is constantly subjected to abrupt salinity changes in the tropical estuaries where it occurs, moving into high salinity areas may be the only way to cope with the impact of L. texanus.     

Developmental stages and molecular phylogeny of Hepatozoon tuatarae, a parasite infecting the New Zealand tuatara, Sphenodon punctatus and the tick, Amblyomma sphenodonti

The developmental stages of Hepatozoon tuatarae were elucidated in both the tuatara host, Sphenodon punctatus and the tick, Amblyomma sphenodonti. PCR amplicons from A. sphenodonti samples identified DNA matching H. tuatarae. Dissection of tick samples showed oogenesis and sporogony occurring in the haemocoel of A. sphenodonti with the average mature oocyst size being 236 × 228 μm. Partial sequence data of the parasite’s small subunit ribosomal gene, obtained by PCR, was used for phylogenetic comparison. Characterisation of the H. tuatarae lifecycle will help in conservation management of the tuatara.     

Sexual recombination punctuated by outbreaks and clonal expansions predicts Toxoplasma gondii population genetics

The cosmopolitan parasitic pathogen Toxoplasma gondii is capable of infecting essentially any warm-blooded vertebrate worldwide, including most birds and mammals, and establishes chronic infections in one-third of the globe’s human population. The success of this highly prevalent zoonosis is largely the result of its ability to propagate both sexually and clonally. Frequent genetic exchanges via sexual recombination among extant parasite lineages that mix in the definitive felid host produces new lines that emerge to expand the parasite’s host range and cause outbreaks. Highly successful lines spread clonally via carnivorism and in some cases sweep to pandemic levels. The extent to which sexual reproduction versus clonal expansion shapes Toxoplasma’s current, global population genetic structure is the central question this review will attempt to answer.     

Protein export in Plasmodium parasites: From the endoplasmic reticulum to the vacuolar export machine

It is somewhat paradoxical that the malaria parasite’s survival strategy involves spending almost all of its blood-stage existence residing behind a two-membrane barrier in a host red blood cell, yet giving considerable attention to exporting parasite-encoded proteins back across these membranes. These exported proteins are thought to play diverse roles and are crucial in pathogenic processes, such as re-modelling of the erythrocyte cytoskeleton and mediating the export of a major virulence protein known as Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1), and in metabolic processes such as nutrient uptake and solute exchange. Despite these varied roles most exported proteins have at least one common link; they share a trafficking pathway that begins with entry into the endoplasmic reticulum and concludes with passage across the vacuole membrane via a proteinaceous translocon known as the Plasmodium translocon of exported proteins (PTEX). In this commentary we review recent advances in our understanding of this export pathway and suggest several models by which different aspects of the process may be interconnected.     

Hematodinium sp. infection of red Paralithodes camtschaticus and blue Paralithodes platypus king crabs from the Sea of Okhotsk, Russia

A disease caused by a parasitic dinoflagellate of the genus Hematodinium was identified in red, Paralithodes camtschaticus, and blue, Paralithodes platypus, king crabs from the north-east region of the Sea of Okhotsk, Russia, during annual stock surveys. No carapace color change was observed even in heavily infected crabs, but diseased crabs possessed creamy-yellow hemolymph, which was visible through the arthrodial membranes of the abdomen and appendages. Several stages of the parasite’s life history, including trophonts, plasmodia, sporonts and macrodinospores, were observed in tissues of infected king crabs. Numerous parasite cells were observed in the lumina of the myocardium, the gills, the connective tissue of antennal glands and the sinuses of nerve ganglia, eyestalks and gastrointestinal tract of king crabs with gross signs of infection. Based on sequencing of the 18S rDNA, it appears that the Hematodinium sp. found in red and blue king crabs is identical or closely related to Hematodinium sp. isolated from crabs of the genera Chionoecetes and Lithodes. Observed prevalences were 0.33% in sublegal male red king crabs, 0.18% in female red king crabs, 0.34% in sublegal male blue king crabs and 0.31% in female blue king crabs.     

Identification of lipids in the cuticle of the parasitic nematode Anisakis simplex and the somatic tissues of the Atlantic cod Gadus morhua

The main aim of this work was to assign the cuticular lipids identified in a parasitic nematode and to distinguish those originating from its host. The hypothesis that long-chained fatty acids and sterols are imported by the parasite in the absence of certain enzymes was also tested. The organisms (Anisakis simplex and Gadus morhua) were extracted in petroleum ether and dichloromethane. Matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF) was used to identify unknown components, and electrospray ionization mass spectrometry (ESI/MS) to verify recognized groups of lipids. The lipid classes identified in the surface layer were free saturated and unsaturated fatty acids, triacylglycerols, sterols and non-polar sphingolipids (ceramides, sphingoid bases). The most abundant fraction consisted of fatty acids. The predominant saturated acids were tetradecanoic acid in the petroleum ether extract of A. simplex, hexadecanoic acid in the dichloromethane extract of A. simplex, and also the polyunsaturated octadecahexaenoic and octadecatrienoic acids in both extracts of the parasitic nematode. The mass spectrum revealed the presence of fatty acids with different numbers of carbons, and with odd and even numbers of unsaturated bonds. The MALDI-TOF mass spectrum also identified triacylglycerols (TAGs). The dominant short-chain TAGs were CoCoCy:₁, CoCoPg and Bu0:0B:₆. The majority of TAGs were found in the ether and dichloromethane extracts of A. simplex. Sterols were the least common class of lipids found in the nematode extracts; most likely, this is the fraction that is entirely incorporated from the host organism because of the parasite’s inability to synthesize them. MALDI-TOF also identified non-polar sphingolipids - ceramides and sphingoid bases. The signals due to N-octanoyl-d-erythro-octasphinganine (m/z 288.3) and N-tetranoyl-d-erythro-tetradecasphinganine (m/z 316.4) were dominant on the mass spectra; quite a large number of short-chain non-polar sphingolipids were also identified.     

Trypanosomatid essential metabolic pathway: New approaches about heme fate in Trypanosoma cruzi

Trypanosoma cruzi, the causal agent of Chagas disease, has a complex life cycle and depends on hosts for its nutritional needs. Our group has investigated heme (Fe-protoporphyrin IX) internalization and the effects on parasite growth, following the fate of this porphyrin in the parasite. Here, we show that epimastigotes cultivated with heme yielded the compounds α-meso-hydroxyheme, verdoheme and biliverdin (as determined by HPLC), suggesting an active heme degradation pathway in this parasite. Furthermore, through immunoprecipitation and immunoblotting assays of epimastigote extracts, we observed recognition by an antibody against mammalian HO-1. We also detected the localization of the HO-1-like protein in the parasite using immunocytochemistry, with antibody staining primarily in the cytoplasm. Although HO has not been described in the parasite’s genome, our results offer new insights into heme metabolism in T. cruzi, revealing potential future therapeutic targets.     

Travellers as sentinels: Assaying the worldwide distribution of polymorphisms associated with artemisinin combination therapy resistance in Plasmodium falciparum using malaria cases imported into Scotland

There is growing evidence that Plasmodium falciparum parasites in southeastern Asia have developed resistance to artemisinin combination therapy. The resistance phenotype has recently been shown to be associated with four single nucleotide polymorphisms in the parasite’s genome. We assessed the prevalence of two of these single nucleotide polymorphisms in P. falciparum parasites imported into Scotland between 2009 and 2012, and in additional field samples from six countries in southeastern Asia. We analysed 28 samples from 11 African countries, and 25 samples from nine countries in Asia/southeastern Asia/Oceania. Single nucleotide polymorphisms associated with artemisinin combination therapy resistance were not observed outside Thailand and Cambodia.     

Studies with the Plasmodium falciparum hexokinase reveal that PfHT limits the rate of glucose entry into glycolysis

To characterise plasmodial glycolysis, we generated two transgenic Plasmodium falciparum lines, one expressing P. falciparum hexokinase (PfHK) tagged with GFP (3D7-PfHK^GFP) and another overexpressing native PfHK (3D7-PfHK⁺). Contrary to previous reports, we propose that PfHK is cytosolic. The glucose analogue, 2-deoxy-d-glucose (2-DG) was nearly 2-fold less toxic to 3D7-PfHK⁺ compared with control parasites, supporting PfHK as a potential drug target. Although PfHK activity was higher in 3D7-PfHK⁺, they accumulated phospho-[¹⁴C]2-DG at the same rate as control parasites. Transgenic parasites overexpressing the parasite’s glucose transporter (PfHT) accumulated phospho-[¹⁴C]2-DG at a higher rate, consistent with glucose transport limiting glucose entry into glycolysis.     

Leishmania infantum chagasi: A genome-based approach to identification of excreted/secreted proteins

The parasitic protozoan, Leishmania, survives in harsh environments within its mammalian and sand fly hosts. Secreted proteins likely play critical roles in the parasite’s interactions with its environment. As a preliminary identification of the spectrum of potential excreted/secreted (ES) proteins of Leishmania infantum chagasi (Lic), a causative agent of visceral leishmaniasis, we used standard algorithms to screen the annotated L. infantum genome for genes whose predicted protein products have an N-terminal signal peptide and lack transmembrane domains and membrane anchors. A suite of 181 candidate ES proteins were identified. These included several that were documented in the literature to be released by other Leishmania spp. Six candidate ES proteins were selected for further validation of their expression and release by different parasite stages. We found both amastigote-specific and promastigote-specific released proteins. The ES proteins of Lic are candidates for future studies of parasite virulence determinants and host protective immunity.     

A standardizable protocol for infection of Rhodnius prolixus with Trypanosoma rangeli, which mimics natural infections and reveals physiological effects of infection upon the insect

Trypanosoma rangeli is a protozoan parasite that shares hosts - mammals and triatomines - with Trypanosoma cruzi, the etiological agent of Chagas disease. Although T. rangeli is customarily considered to be non-pathogenic to human hosts, it is able to produce pathologies in its invertebrate hosts. However, advances are hindered by a lack of standardization of infection procedures and these pathologies need documentation. To establish a suitable, and standardizable, infection protocol, the duration of the fourth instar was evaluated in nymphs infected by injection into the thorax with different concentrations of parasites, and compared with nymphs infected naturally (i.e. orally). We demonstrate that delays in moult were attributable to the presence of the parasite in the haemolymph (vs. the gut) and propose that the protocol presented here simulates closely natural infections. This methodology was then used for the evaluation of physiological parameters and several hitherto unreported effects of T. rangeli infection on Rhodnius prolixus were revealed. Haemolymph volume was greater in infected than uninfected nymphs but this alteration could not be attributed to water retention, since infected insects lost the same amount of water as controls. However, we found that lipid content and fat body weight were both increased in insects infected by T. rangeli. We propose that this is due to the parasite’s sequestration of host blood lipids and carrier proteins. With these findings, we have taken a few first steps to unravelling physiological details of the host-parasite interaction. We suggest future directions towards a fuller understanding of mechanistic and adaptive aspects of triatomine-trypanosomatid interactions.     

Fasciola gigantica: Histology of the digestive tract and the expression of cathepsin L

The digestive tract of Fasciola gigantica is composed of the oral sucker, buccal tube, pharynx, esophagus, and caecum. The tegumental-type epithelium lines the first four parts of the digestive tract while the caecal-type epithelium lines the remaining parts from the caecal bifurcation. The caecal-epithelial cells are classified into 3 types according to their staining properties and ultrastructural characteristics, as related to the amount of food contents in the caecal lumen. All caecal-type epithelial cells synthesize and secrete cathepsin L, a major group of enzymes in the digestive tract, as detected by in situ hybridization and immunolocalization. Moreover, the secreted cathepsin L is also adsorbed on the outer surface of the tegument and the glycocalyx coating of the surface of the tegument, whereas the tegumental cells and tegumental syncytium covering the parasite’s body and lining the proximal part of the digestive tract exhibit no in situ hybridization signal and immunostaining for cathepsin L.     

Gene silencing reveals a crucial role for anti-lipopolysaccharide factors from Penaeus monodon in the protection against microbial infections

Anti-lipopolysaccharide factors (ALFs) are antimicrobial peptides previously identified in various crustaceans. Out of five isoforms identified in Penaeus monodon, ALFPm3 is the best characterized, exhibits antibacterial and antifungal activities and can protect the shrimp from viral infections. Herein, the most recent identified ALFPm, called ALFPm6, is characterized for its potential role in the shrimp’s immunity. RNA interference-mediated gene silencing was used to study the function of ALFPm6 in comparison to ALFPm3. Knockdown of ALFPm3 gene led to rapid death with a cumulative shrimp mortality of 86% within 7 days, accompanied by a 12- and 50-fold higher bacterial count after 2 days in the haemolymph and hepatopancreas, respectively, compared to the control shrimp injected with GFP dsRNA. In contrast, gene silencing of ALFPm6 alone had no effect on the shrimp mortality, but led to a significant increase in the cumulative mortality and a faster mortality rate following Vibrio harveyi and white spot syndrome virus (WSSV) infections, respectively. These results support the roles of ALFPm6 and ALFPm3 in the protection of shrimp against microbial infections.     

Localisation of three host-protective oncospheral antigens of Taenia ovis

Immunohistochemistry, confocal immunofluorescence and immunogold labelling were used to determine the localisation of the host-protective antigens To16, To18 and To45W in Taenia ovis oncospheres. During maturation of the adult tapeworm the antigens were initially seen as diffuse staining in the developing oncospheres but in mature oncospheres four distinct cells stained positively for the antigens. Confocal fluorescence microscopy using different fluorophores revealed that each of the antigens co-localises within the same cells in the oncosphere. No surface localisation was seen in non-activated or recently activated parasites. Immunogold labelling of non-activated oncosphere sections viewed in transmission electron microscopy revealed labelling of bilateral cells, however the identities of these cells was unclear due to deficiencies in the current level of understanding of oncosphere ultrastructure. Localisation of all the antigens changed dramatically after oncospheres were activated in vitro with each of the antigens being dispersed more generally throughout the parasite parenchyma. During development of the parasites in in vitro culture, surface localisation of the proteins was seen in parasites after 3 or more days in culture. All three antigens were found to be completely absent in parasites by 15 days of culture. The location of the host-protective antigens suggests that initially the invading oncospheres are not susceptible to vaccine-induced antibody and complement mediated attack, but that as the parasites mature, the host-protective antigens come to be associated with the parasite’s surface, rendering them susceptible to immune attack.     

Hepatopancreas but not ovary is the site of vitellogenin synthesis in female fresh water crab, Oziothelphusa senex senex

The objective of the present study was to explore the site of synthesis of vitellogenin (Vtg) in fresh water edible crab, Oziothelphusa senex senex. Vtg cDNA fragments were isolated from the hepatopancreas of female crabs using RT-PCR method, and the deduced amino acid sequence of O. senex senex showed more than 60% identity with other brachyuran Vtg sequences. RT-PCR analysis showed that Vtg mRNA can be detected only in hepatopancreas of female Oziothelphusa but not in other tissues including eyestalks, Y-organs, mandibular organs, thoracic ganglion, hypodermis and ovary. Antibodies were raised against vitellin purified from the ovary of O. senex senex. Immunoprecipitation analysis revealed the presence of Vtg in the hepatopancreas of vitellogenic stage I females and in the hemolymph, hepatopancreas and ovary extracts from vitellogenic stage II females but absent in hemolymph and hepatopancreas extract of males. These results suggest that Vtg is synthesized only in hepatopancreas but not in the ovaries of O. senex senex. In addition, Vtg synthesized in hepatopancreas is transported to ovary through hemolymph.     

Effects of intermediate host genetic background on parasite transmission dynamics: a case study using Schistosoma mansoni

For parasites that require multiple hosts to complete their development, genetic interplay with one host may impact parasite transmission and establishment in subsequent hosts. In this study, we used microsatellite loci to address whether the genetic background of snail intermediate hosts influences life-history traits and transmission patterns of dioecious trematode parasites in their definitive hosts. We performed experimental Schistosoma mansoni infections utilizing two allopatric populations of Biomphalaria glabrata snails and assessed intensities and sex ratios of adult parasites in mouse definitive hosts. Our results suggest that the genetic background of hosts at one point in a parasite’s life cycle can influence the intensities and sex ratios of worms in subsequent hosts.