Biology of Cryptosporidium from marsupial hosts

The majority of biological data on Cryptosporidium has been collected from humans and domestic animal hosts which creates a bias in knowledge on the biodiversity and evolution of this parasite genus. Further to understanding Cryptosporidium biology are studies encompassing broad hosts that represent diverse taxa sampled across wide geographic ranges. Marsupials represent a group of wildlife hosts from which limited information on Cryptosporidium is available. As marsupial hosts are an ancient mammalian lineage they represent an important group for studying parasite evolution. This review summarises information of the biology, epidemiology and evolution of Cryptosporidium in marsupial hosts, and discusses the importance of further understanding interactions in this parasite-host system.     

Within-host dynamics of Trichinella spiralis predict persistent parasite transmission in rat populations

Trichinella spiralis is transmitted and maintained in both a domestic and sylvatic cycle, whereby rats contribute to the spread of T. spiralis from domestic to sylvatic animals and vice versa. As a model for T. spiralis transmission in wildlife, we studied the potential of rats to act as a reservoir species for T. spiralis, by assessing experimentally its within-host infection dynamics, and simulating the between-host dynamics by a Monte Carlo approach. The distribution of parasite burden in individual rats is mathematically defined by roots of the dose response equation intersecting with the diagonal. In simulated between-host dynamics, up to 10⁴ events of uninterrupted parasite transmission were observed. Histograms of parasite burdens per individual rat matched closely with the mixture of two gamma distributions, which were derived from the within-host infection dynamics. In conclusion, T. spiralis transmission persists in a population of rats when they cannibalize their own species. Rats should be included in the minimal set of wildlife species that maintain the life cycle of T. spiralis.     

A multiplex PCR that discriminates between Trypanosoma brucei brucei and zoonotic T. b. rhodesiense

Two subspecies of Trypanosoma brucei s.l. co-exist within the animal populations of Eastern Africa; T. b. brucei a parasite which only infects livestock and wildlife and T. b. rhodesiense a zoonotic parasite which infects domestic livestock, wildlife, and which in humans, results in the disease known as Human African Trypanosomiasis (HAT) or sleeping sickness. In order to assess the risk posed to humans from HAT it is necessary to identify animals harbouring potentially human infective parasites. The multiplex PCR method described here permits differentiation of human and non-human infective parasites T. b. rhodesiense and T. b. brucei based on the presence or absence of the SRA gene (specific for East African T. b. rhodesiense), inclusion of GPI-PLC as an internal control indicates whether sufficient genomic material is present for detection of a single copy T. brucei gene in the PCR reaction.     

Successful elimination of a lethal wildlife infectious disease in nature

Methods to mitigate the impacts of emerging infectious diseases affecting wildlife are urgently needed to combat loss of biodiversity. However, the successful mitigation of wildlife pathogens in situ has rarely occurred. Indeed, most strategies for combating wildlife diseases remain theoretical, despite the wealth of information available for combating infections in livestock and crops. Here, we report the outcome of a 5-year effort to eliminate infection with Batrachochytrium dendrobatidis affecting an island system with a single amphibian host. Our initial efforts to eliminate infection in the larval reservoir using a direct application of an antifungal were successful ex situ but infection returned to previous levels when tadpoles with cleared infections were returned to their natal sites. We subsequently combined antifungal treatment of tadpoles with environmental chemical disinfection. Infection at four of the five pools where infection had previously been recorded was eradicated, and remained so for 2 years post-application.     

The identification of a new Giardia duodenalis assemblage in marine vertebrates and a preliminary analysis of G. duodenalis population biology in marine systems

Giardia duodenalis is an intestinal parasite of many vertebrates. The presence of G. duodenalis in the marine environment due to anthropogenic and wildlife activity is well documented, including the contributions from untreated sewage and storm water, agricultural run-off and droppings from terrestrial animals. Recently, studies have detected this protistan parasite in the faeces of marine vertebrates such as whales, dolphins, seals and shore birds. To explore the population biology of G. duodenalis in marine life, we determined the prevalence of G. duodenalis in two species of seal (Halichoerus grypus, Phoca vitulina vitulina and Phoca vitulina richardsi) from the east and west coasts of the USA, sequenced two loci from G. duodenalis-positive samples to assess molecular diversity and examined G. duodenalis distribution amongst these seals and other marine vertebrates along the east coast. We found a significant difference in the presence of G. duodenalis between east and west coast seal species. Only the zoonotic lineages of G. duodenalis, Assemblages A and B and a novel lineage, which we designated as Assemblage H, were identified in marine vertebrates. Assemblages A and B are broadly distributed geographically and show a lack of host specificity. Only grey seal (Halichoerus grypus) samples and one gull sample (Larus argentatus) from a northern location of Cape Cod, Massachusetts, USA, showed the presence of Assemblage H haplotypes; only one other study of harbour seals from the Puget Sound region of Washington, USA previously recorded the presence of an Assemblage H haplotype. Assemblage H sequences form a monophyletic clade that appears as divergent from the other seven Assemblages of G. duodenalis as these assemblages are from each other. The discovery of a previously uncharacterised lineage of G. duodenalis suggests that this parasite has more genetic diversity and perhaps a larger host range than previously believed.     

Presidential address: rediscovering parasites using molecular tools--towards revising the taxonomy of Echinococcus, Giardia and Cryptosporidium

Molecular biology has provided parasitologists with a fantastic variety of techniques that have had a major impact on research into parasites and parasitism. Molecular tools have revealed the extent and nature of genetic diversity in parasites and this information has made a significant contribution to studies on the population genetics and evolutionary biology of parasites. Similarly, epidemiology has benefited enormously from the application of molecular tools in terms of studying parasite life cycles and transmission, and in the development of specific and sensitive methods for diagnosis and surveillance. However, the theme I wish to develop in this paper is concerned with the contribution molecular tools have made to parasite taxonomy and systematics, and in particular, the fact that in many cases molecular tools are validating the proposals made many years ago by taxonomists and biologists which were discounted or not fully accepted at the time. To do this I have chosen four examples (Echinococcus, Entamoeba, Giardia, Cryptosporidium) where recent research involving molecular characterisation has confirmed observations made many years ago and has resulted in a need to revise the taxonomy of different groups of parasites.     

Resistance to antiparasitic drugs: the role of molecular diagnosis

Chemotherapy is central to the control of many parasite infections of both medical and veterinary importance. However, control has been compromised by the emergence of drug resistance in several important parasite species. Such parasites cover a broad phylogenetic range and include protozoa, helminths and arthropods. In order to achieve effective parasite control in the future, the recognition and diagnosis of resistance will be crucial. This demand for early, accurate diagnosis of resistance to specific drugs in different parasite species can potentially be met by modern molecular techniques. This paper summarises the resistance status of a range of important parasites and reviews the available molecular techniques for resistance diagnosis. Opportunities for applying successes in some species to other species where resistance is less well understood are explored. The practical application of molecular techniques and the impact of the technology on improving parasite control are discussed.     

Interspecific antagonism and virulence in hosts exposed to two parasite species

Co-infection of host organisms by multiple parasite species has evolutionary consequences for all participants in the symbiosis. In this study, we co-exposed aquatic-snails (Biomphalaria glabrata) to two of their trematode parasites, Schistosoma mansoni and Echinostoma caproni. In co-exposed snails, E. caproni prevalence was 63% compared to only 23% for S. mansoni. Co-exposed E. caproni-infected snails exhibited reduced fecundity, higher mortality, and higher parasite reproduction (higher virulence) compared to hosts exposed to echinostomes alone. Conversely, co-exposed S. mansoni-infected snails released fewer parasites and produced greater numbers of eggs compared to hosts exposed to S. mansoni alone. These results suggest that co-exposure not only influences the establishment (presence or absence) of particular parasite species, but also impacts host life history, parasite reproduction, and the virulence of the interaction.     

Parasite age-intensity relationships in red-spotted newts: does immune memory influence salamander disease dynamics?

Acquired immune memory in vertebrates influences transmission and persistence of infections, with consequences for parasite dynamics at both the individual and population levels. The potential impact of acquired immunity is of particular interest for salamanders, whose acquired immune systems are thought to be less effective than those of frogs and other tetrapods. One way to examine the importance of acquired immunity to parasite dynamics at the population level is by examining the relationship between host age and parasite infection intensity. Acquired immunity reduces infection rates in older animals, causing decreased parasite intensity in older age classes and leading to curvilinear age-intensity relationships for persistent parasites and convex age-intensity relationships for transient parasites. We used age-intensity relationships to look for the signature of acquired immunity for 12 parasite taxa of red-spotted newts (Notophthalmus viridescens), using data from a 2-year parasitological survey of six newt populations. We estimated ages from snout-vent length (SVL) based on the relationship between SVL and skeletochronologically-derived ages in a subset of newts. We found evidence of acquired immunity to two parasite taxa, bacterial pathogens and the protist Amphibiocystidium viridescens, whose convex age-intensity relationships could not be easily explained by alternative mechanisms. Our results suggest that the acquired immune response of newts is sufficient to influence the dynamics of at least some parasites.     

Hidden diversity and evolutionary trends in malacosporean parasites (Cnidaria: Myxozoa) identified using molecular phylogenetics

Malacosporeans represent a small fraction of myxozoan biodiversity with only two genera and three species described. They cycle between bryozoans and freshwater fish. In this study, we (i) microscopically examine and screen different freshwater/marine fish species from various geographic locations and habitats for the presence of malacosporeans using PCR; (ii) study the morphology, prevalence, host species/habitat preference and distribution of malacosporeans; (iii) perform small subunit/large subunit rDNA and Elongation factor 2 based phylogenetic analyses of newly gathered data, together with all available malacosporean data in GenBank; and (iv) investigate the evolutionary trends of malacosporeans by mapping the morphology of bryozoan-related stages, host species, habitat and geographic data on the small subunit rDNA-based phylogenetic tree. We reveal a high prevalence and diversity of malacosporeans in several fish hosts in European freshwater habitats by adding five new species of Buddenbrockia and Tetracapsuloides from cyprinid and perciform fishes. Comprehensive phylogenetic analyses revealed that, apart from Buddenbrockia and Tetracapsuloides clades, a novel malacosporean lineage (likely a new genus) exists. The fish host species spectrum was extended for Buddenbrockia plumatellae and Buddenbrockia sp. 2. Co-infections of up to three malacosporean species were found in individual fish. The significant increase in malacosporean species richness revealed in the present study points to a hidden biodiversity in this parasite group. This is most probably due to the cryptic nature of malacosporean sporogonic and presporogonic stages and mostly asymptomatic infections in the fish hosts. The potential existence of malacosporean life cycles in the marine environment as well as the evolution of worm- and sac-like morphology is discussed. This study improves the understanding of the biodiversity, prevalence, distribution, habitat and host preference of malacosporeans and unveils their evolutionary trends.     

Transmission and burden and the impact of temperature on two species of vertically transmitted microsporidia

Microsporidia are unusual amongst eukaryotic parasites in that they utilize both vertical and horizontal transmission and vertically transmitted species can cause sex ratio distortion in their host. Here we study vertical transmission in two species of feminising microsporidia, Nosema granulosis and Dictyocoela duebenum, infecting a single population of the crustacean host Gammarus duebeni and measure the effect of temperature on parasite transmission and replication. N. granulosis was vertically transmitted to 82% of the host embryos and D. duebenum was transmitted to 72% of host embryos. For both parasites, we report relatively low parasite burdens in developing host embryos. However, the parasites differ in their pattern of replication and burden within developing embryos. Whilst N. granulosis undergoes replication during host development, the burden of D. duebenum declines, leading us to propose that parasite dosage and feminisation efficiency underlie the different parasite frequencies in the field. We also examine the effect of temperature on parasite transmission and replication. Temperature does not affect the percentage of young that inherit the infection. However, low temperatures inhibit parasite replication relative to host cell division, resulting in a reduction in parasite burden in infected embryos. The reduced parasite burden at low temperatures may underpin reduced feminization at low temperatures and so limit the spread of sex ratio distorters through the host population.     

Investigating the possible role of benthic macroinvertebrates and zooplankton in the life cycle of the haplosporidian Bonamia ostreae

Bonamia ostreae is a protistan parasite of the European flat oyster, Ostrea edulis. Though direct transmission of the parasite can occur between oysters, it is unclear if this represents the complete life cycle of the parasite, and the role of a secondary or intermediate host or carrier species cannot be ruled out. In this preliminary study, benthic macroinvertebrates and zooplankton from a B. ostreae-endemic area were screened for the presence of parasite DNA, using polymerase chain reaction (PCR). Eight benthic macroinvertebrates and nineteen grouped zooplankton samples gave positive results. Certain species, found positive for the parasite DNA, were then used in laboratory transmission trials, to investigate if they could infect na?ve oysters. Transmission of B. ostreae was effected to two na?ve oysters cohabiting with the brittle star, Ophiothrix fragilis.     

New evidence for the involvement of Paracartia grani (Copepoda,Calanoida) in the life cycle of Marteilia refringens (Paramyxea)

The dynamics of the protozoan parasite Marteilia refringens was studied in Thau lagoon, an important French shellfish site, for 1 year in three potential hosts: the Mediterranean mussel Mytilus galloprovincialis (Mytiliidae), the grooved carpet shell Ruditapes decussatus (Veneriidae) and the copepod Paracartia grani (Acartiidae). Parasite DNA was detected by PCR in R. decussatus. In situ hybridisation showed necrotic cells of M. refringens in the digestive epithelia of some R. decussatus suggesting the non-involvement of this species in the parasite life cycle. In contrast, the detection of M. refringens in mussels using PCR appeared bimodal with two peaks in spring and autumn. Histological observations of PCR-positive mussels revealed the presence of different parasite stages including mature sporangia in spring and autumn. These results suggest that the parasite has two cycles per year in the Thau lagoon and that mussels release parasites into the water column during these two periods. Moreover, PCR detection of the parasite in the copepodid stages of P. grani between June and November supports the hypothesis of the transmission of the parasite from mussels to copepods and conversely. In situ hybridisation performed on copepodites showed labeling in some sections. Unusual M. refringens cells were observed in the digestive tract and the gonad from the third copepodid stage, suggesting that the parasite could infect a copepod by ingestion and be released through the gonad. This hypothesis is supported by the PCR detection of parasite DNA in copepod eggs from PCR-positive females, which suggests that eggs could contribute to the parasite spreading in the water and could allow overwintering of M. refringens. Finally, in order to understand the interactions between mussels and copepods, mussel retention efficiency (number of copepods retained by a mussel) was measured for all P. grani developmental stages. Results showed that all copepod stages could contribute to the transmission of the parasite, especially eggs and nauplii which were retained by up to 90%.     

Antiretroviral protease inhibitors potentiate chloroquine antimalarial activity in malaria parasites by regulating intracellular glutathione metabolism

Antiretroviral protease inhibitors significantly potentiated the sensitivity of chloroquine-resistant malaria parasites to the antimalarial drug in vitro and in vivo. Ritonavir was found to be potent in potentiating CQ antimalarial activities in both -resistant and -sensitive lines. The mechanism by which the APIs modulate the CQ resistance in malaria parasites was further investigated. CQ-resistant parasites showed increased intracellular glutathione levels in comparison with the CQ-sensitive parasites. Treatment with APIs significantly reduced the levels of GSH and glutathione S-transferase activities in CQ-resistant parasites. Ritonavir also decreased glutathione reductase activities and glutathione peroxidase activities in CQ-resistant parasite line. Taken together, these results demonstrate that parasite GSH and GST may play an important role in CQ resistance and APIs are able to enhance the sensitivity of CQ-resistant malaria parasite to the drug by influencing the levels of GSH and the activities of the related enzymes.     

Invasion of Ceratomyxa shasta (Myxozoa) and comparison of migration to the intestine between susceptible and resistant fish hosts

The myxozoan parasite Ceratomyxa shasta infects salmonids causing ceratomyxosis, a disease elicited by proliferation of the parasite in the intestine. This parasite is endemic to the Pacific Northwest of North America and salmon and trout strains from endemic river basins show increased resistance to the parasite. It has been suggested that these resistant fish (i) exclude the parasite at the site of invasion and/or (ii) prevent establishment in the intestine. Using parasites pre-labeled with a fluorescent stain, carboxyfluorescein succinimidyl diacetate (CFSE), the gills were identified as the site of attachment of C. shasta in a susceptible fish strain. In situ hybridization (ISH) of histological sections was then used to describe the invasion of the parasites in the gill filaments. To investigate differences in the progress of infection between resistant and susceptible fish, a C. shasta-susceptible strain of rainbow trout (Oncorhynchus mykiss) and a C. shasta-resistant strain of Chinook salmon (Oncorhynchus tshawytscha) were sampled at consecutive time points following exposure at an endemic site. Using ISH in both species, the parasite was observed to migrate from the gill epithelium into the gill blood vessels where replication and release of parasite stages occurred. Quantitative PCR verified entry of the parasite into the blood. Parasite levels in blood increased 4 days p.i. and remained at a consistent level until the second week when parasite abundance increased further and coincided with host mortality. The timing of parasite replication and migration to the intestine were similar for both fish species. The field exposure dose was unexpectedly high and apparently overwhelmed the Chinook salmon’s defenses, as no evidence of resistance to parasite penetration into the gills or prevention of parasite establishment in the intestine was observed.     

Cyclin-dependent kinase homologues of Plasmodium falciparum

The intraerythrocytic asexual cycle of the malarial parasite is complex and atypical: during schizogony the parasite undergoes multiple rounds of DNA replication and asynchronous nuclear division without cytokinesis. This cell cycle deviates from the classical eukaryotic cell cycle model where, 'DNA replicates only once per cell cycle'. A clear understanding of the molecular switches that control this unusual developmental cycle would be of great interest, both in terms of fundamental Plasmodium biology and in terms of novel potential drug target identification. In recent years considerable effort has been made to identify the malarial orthologues of the cyclin-dependent kinases, which are key regulators of the orderly progression of the eukaryotic cell cycle. This review focuses on the current state-of-knowledge of Plasmodium falciparum cyclin-dependent kinase-like kinases and their regulators.     

Susceptibility of juvenile Crassostrea gigas and resistance of Panope abrupta to Mikrocytos mackini

Samples from the field and laboratory exposure to Mikrocytos mackini (a tiny protistan parasite of unknown taxonomic affiliation) confirmed that juvenile Pacific oysters (Crassostrea gigas) are susceptible to infection and the resulting disease. In the laboratory bath exposure experiment, a prevalence of infection approaching 100% and mortalities were observed in the small oysters (about 18 mm in shell length). However, in the same laboratory exposure experiment, similar aged geoduck clams (Panope abrupta, about 8mm in shell length) were resistant to infection. The main route of infection in the oysters appeared to be via the digestive tract and possibly the gills where the parasite multiplied within host cells. Other tissues such as the adductor muscle and vesicular connective tissue were subsequently colonized. Although the infection resulted in the mortality of some oysters, others appeared to overcome the disease.     

Early removal of alternatively activated macrophages leads to Taenia crassiceps cysticercosis clearance in vivo

To determine the role of alternatively activated macrophages in modulating the outcome of experimental cysticercosis caused by Taenia crassiceps, we investigated the effect of removal of alternatively activated macrophage by injecting clodronate-loaded liposomes into susceptible BALB/c mice. Following T. crassiceps infection, mice receiving PBS-loaded liposomes developed a dominant Th2-type response associated with the presence of alternatively activated macrophages together with antigen-specific hyporesponsiveness and high parasite burden. In contrast, similarly infected mice treated with clodronate-loaded liposomes mounted a mixed Th1/Th2-type response, reversed antigen-specific hyporesponsiveness and did not carry notable alternatively activated macrophage populations. These factors were associated with increased resistance to T. crassiceps cysticercosis. Interestingly, early AAM? depletion was enough to limit parasite growth. However, if macrophages were depleted late in the infection, no effect on parasite burden was observed. These findings demonstrate that alternatively activated macrophages play a critical role in mediating susceptibility to experimental cysticercosis in which their early recruitment may favor parasite survival.     

Dense granules: are they key organelles to help understand the parasitophorous vacuole of all apicomplexa parasites?

Together with micronemes and rhoptries, dense granules are specialised secretory organelles of Apicomplexa parasites. Among Apicomplexa, Plasmodium represents a model of parasites propagated by way of an insect vector, whereas Toxoplasma is a model of food borne protozoa forming cysts. Through comparison of both models, this review summarises data accumulated over recent years on alternative strategies chosen by these parasites to develop within a parasitophorous vacuole and explores the role of dense granules in this process. One of the characteristics of the Plasmodium erythrocyte stages is to export numerous parasite proteins into both the host cell cytoplasm and/or plasma membrane via the vacuole used as a step trafficking compartment. Whether this feature can be correlated to few storage granules and a restricted number of dense granule proteins, is not yet clear. By contrast, the Toxoplasma developing vacuole is decorated by abundantly expressed dense granule proteins and is characterised by a network of membranous nanotubes. Although the exact function of most of these proteins remains currently unknown, recent data suggest that some of these dense granule proteins could be involved in building the intravacuolar membranous network. Conserved expression of the Toxoplasma dense granule proteins throughout most of the parasite stages suggests that they could also be key elements of the cyst formation.     

An example of molecular co-evolution: reactive oxygen species (ROS) and ROS scavenger levels in Schistosoma mansoni/Biomphalaria glabrata interactions

The co-evolution between hosts and parasites involves huge reciprocal selective pressures on both protagonists. However, relatively few reports have evaluated the impact of these reciprocal pressures on the molecular determinants at the core of the relevant interaction, such as the factors influencing parasitic virulence and host resistance. Here, we address this question in a host-parasite model that allows co-evolution to be monitored in the field: the interaction between the mollusc, Biomphalaria glabrata, and its trematode parasite, Schistosoma mansoni. Reactive oxygen species (ROS) produced by the haemocytes of B. glabrata are known to play a crucial role in killing S. mansoni. Therefore, the parasite must defend itself against oxidative damage caused by ROS using ROS scavengers in order to survive. In this context, ROS and ROS scavengers are involved in a co-evolutionary arms race, and their respective production levels by sympatric host and parasite could be expected to be closely related. Here, we test this hypothesis by comparing host oxidant and parasite antioxidant capabilities between two S. mansoni/B. glabrata populations that have co-evolved independently. As expected, our findings show a clear link between the oxidant and antioxidant levels, presumably resulting from sympatric co-evolution. We believe this work provides the first supporting evidence of the Red Queen Hypothesis of reciprocal evolution for functional traits at the field-level in a model involving a host and a eukaryotic parasite.