Prospects for exploring molecular developmental processes in Haemonchus contortus

Haemonchus contortus of small ruminants is a parasitic nematode of major socio-economic importance world-wide. While there is considerable knowledge of the morphological changes which take place during the life cycle of H. contortus, very little is understood about the molecular and biochemical processes which govern developmental changes in the parasite. Recent technological advances and the imminent genomic sequence for H. contortus provide unique opportunities to investigate the molecular basis of such processes in parasitic nematodes. This article reviews molecular and biochemical aspects of development in H. contortus, reports on some recent progress on signal transduction molecules in this parasite and emphasises the opportunities that new technologies and the free-living nematode, Caenorhabditis elegans, offer for investigating developmental aspects in H. contortus and related strongylid nematodes, also in relation to developing novel approaches for control.     

Development of a vaccine against haemonchus contortus

Haemonchus contortus is an economically important nematode parasite of sheep and the occurrence worldwide of strains resistant to anthelmintic chemicals has emphasized the need to develop a vaccine against it. Here, Ed Munn describes the approach to this problem adopted in his laboratory. The principle developed should be applicable to other gastrointestinal parasites.     

Haemonchus contortus: in vitro drug screening assays with the adult life stage

Motility and feeding assays were assessed as in vitro systems for screening of novel compounds for anthelmintic activity against adult Haemonchus contortus. The study aimed to develop an assay with the parasitic adult stage of this species that could be used in conjunction with, or as an alternative to, the free-living larval stage screens commonly used for drug discovery with many parasitic nematode species. The feeding assay showed limitations due to the apparent continuation of a significant degree of feeding in worms showing greatly reduced motility in the presence of some drugs. Hence, it appeared most likely that the feeding assay would underestimate the toxicity of these drugs. The motility assay was able to detect toxicity of known anthelmintics, including the 'slow-acting' benzimidazoles. A small-scale screening exercise used the motility assay to detect toxicity towards adult parasites in 10 compounds out of a group of 200 chemicals (selected due to known toxic effects in larval development assays). The motility assay appeared suitable for drug screening against adult H. contortus. The use of the adult stage for drug screening in this way ensures that the drug is toxic towards the parasite life stage to be targeted in vivo. A lack of activity in subsequent in vivo trials could, therefore, be most likely attributable to host pharmacokinetic factors rather than an intrinsic lack of activity of the drug towards the adult parasite.     

Evaluation of Caenorhabditis elegans glycoproteins as protective immunogens against Haemonchus contortus challenge in sheep

High levels of protection can be attained against Haemonchus contortus challenge infection in sheep using native antigens isolated from the gut of the adult parasite. However, vaccination with recombinant forms of these antigens, or components thereof, has disappointingly failed to generate similar levels of protection, suggesting that appropriate nematode glycosylation may be a prerequisite for protection. The free-living nematode, Caenorhabditis elegans is closely related to H. contortus and has been shown to share similar glycan moieties. In order to investigate the potentially protective role of these glycan moieties, a complex set of glycoproteins was isolated from C. elegans using ConA-lectin chromatography and their efficacy as immunogens against H. contortus challenge infection evaluated in sheep. Despite the generation of a high titre systemic IgG antibody response to the C. elegans glycoproteins and the ability of these antibodies to bind to the microvillar surface of the gut of H. contortus, no protection against challenge infection was observed. Serum antibodies to the C. elegans glycoproteins cross-reacted with the H. contortus host-protective antigen, H-gal-GP, by ELISA, although the level of cross-reactivity was not of a magnitude considered protective. Qualitative differences were also determined between the glycan epitopes of the C. elegans ConA-binding proteins and those of H-gal-GP, suggesting the presence of H. contortus-specific patterns of glycosylation.     

Ultrastructural study on a novel microsporidian, Endoreticulatus eriocheir sp. nov. (Microsporidia, Encephalitozoonidae), parasite of Chinese mitten crab, Eriocheir sinensis (Crustacea, Decapoda)

A microsporidian pathogen, infecting the epithelial cells of the hepatopancreas of Chinese mitten crab, Eriocheir sinensis, was studied by electron microscopy. The detailed ultrastructure of life cycle of the pathogen including proliferative and sporogonic developmental stages are provided. All stages of the parasite are haplokaryotic and develop in a vacuole bounded by a single membrane in contact with host cell cytoplasm. Sporogenesis is synchronous with the same developmental stage in one vacuole. Sporogony shows a characteristic of multinucleate sporogonial plasmodia divided by rosette-like division, producing 4 or 8 sporoblasts. The mature spore is ellipsoidal, length (mean) 1.7 microm, width 1.0 microm, with a uninucleate in the center of the sporoplasm, 7 turns of the polar filament, a bell-like polaroplast of compact membranes and obliquely positioned posterior vacuole. The morphological characteristics of this novel microsporidian pathogen have led us to assign the parasite to a new species of Endoreticulatus, E. eriocheir sp. nov., that has not been reported previously from crab.     

Parasite-mediated selection drives an immunogenetic trade-off in plains zebras (Equus quagga)

Pathogen evasion of the host immune system is a key force driving extreme polymorphism in genes of the major histocompatibility complex (MHC). Although this gene family is well characterized in structure and function, there is still much debate surrounding the mechanisms by which MHC diversity is selectively maintained. Many studies have investigated relationships between MHC variation and specific pathogens, and have found mixed support for and against the hypotheses of heterozygote advantage, frequency-dependent or fluctuating selection. Few, however, have focused on the selective effects of multiple parasite types on host immunogenetic patterns. Here, we examined relationships between variation in the equine MHC gene, ELA-DRA, and both gastrointestinal (GI) and ectoparasitism in plains zebras (Equus quagga). Specific alleles present at opposing population frequencies had antagonistic effects, with rare alleles associated with increased GI parasitism and common alleles with increased tick burdens. These results support a frequency-dependent mechanism, but are also consistent with fluctuating selection. Maladaptive GI parasite ‘susceptibility alleles’ were reduced in frequency, suggesting that these parasites may play a greater selective role at this locus. Heterozygote advantage, in terms of allele mutational divergence, also predicted decreased GI parasite burden in genotypes with a common allele. We conclude that an immunogenetic trade-off affects resistance/susceptibility to parasites in this system. Because GI and ectoparasites do not directly interact within hosts, our results uniquely show that antagonistic parasite interactions can be indirectly modulated through the host immune system. This study highlights the importance of investigating the role of multiple parasites in shaping patterns of host immunogenetic variation.     

Growth and development of Haemonchus contortus in jirds, Meriones unguiculatus.

Growth and development of Haemonchus contortus were examined in jirds and were compared to these processes in lambs. Number, sex, size, and stage of development were determined for worms recovered at necropsy at various times postinoculation (PI) from immunosuppressed jirds inoculated with approximately 1,000 exsheathed infective larvae (L3) of H. contortus. In addition, gastric tissue samples from jirds were examined histologically. Parallel studies were done in lambs inoculated with approximately 7,500 L3. Typically, 5-30% of the inoculum established and survived in jirds at reasonably stable numbers to day 14 PI. By day 21 PI, worm numbers in jirds decreased dramatically. Although the parasite was similar in size and development on day 4 PI in jirds and lambs, from day 7 PI on, worms were significantly smaller and less developed in jirds. On histological examination, the parasite was found only in the glandular portion of the stomach of jirds (anatomically similar to its predilection site in the abomasum of lambs), and histological changes were consistent for both host species. Although growth and development of H. contortus are slower and incomplete in jirds, the parasite establishes, grows, and develops (at anatomically comparable sites in both hosts) in this model. Thus, the model appears to provide a useful laboratory host to study H. contortus.     

Trends and challenges in the effective and sustainable control of Haemonchus contortus infection in sheep. Review

Haemonchosis, with its very wide distribution, has become a very important production constraint in sheep farms in tropical, subtropical and temperate regions worldwide. Various intrinsic and extrinsic factors determine the survival of Haemonchus contortus and hence the development of the disease in the animal. In general, control of gastrointestinal nematode infestation in sheep relies heavily on anthelmintic treatments. However, the indiscriminate use of these drugs has led to the widespread emergence of drug resistant strains of parasites, that has necessitated the development and use of various parasite control methods such as grazing management, biological agents and vaccines and the selection of resistant breeds of animals, with or without moderate use of anthelmintics. The ultimate goal of such control programs is to enhance productivity, while minimising risks regarding drug resistance and consumer and environmental concerns. This review attempts to highlight the different methods employed in the control of haemonchosis in sheep and the practical limitations associated with both control programs and the internal and external factors associated with the parasite and its microenvironment.     

Infection of immunosuppressed mice with the abomasal nematode parasite of ruminants, Haemonchus contortus

Infective larvae of Haemonchus contortus established in mice either immunosuppressed with the corticosteroid, dexamethasone, or the cytotoxic drug, cyclophosphamide, or treated with the histamine H2 inhibitor, cimetidine. Infections persisted for as long as the immunosuppressive treatment (7 days) and growth of larvae was similar to that seen in sheep. Virtually no larvae survived in untreated mice. Accordingly, it would appear that adaptive immunity is an important barrier against primary infection by H. contortus in mice and is a determinant of host-range for this parasite. Antibody raised in either sheep or mice against soluble extracts of adult H. contortus precipitated with different but overlapping sets of worm antigens. This suggests that the unique antigens recognized by the mouse compared with the sheep are crucial for the rapid protective responses which prevent primary infection.     

No adaptation of Haemonchus contortus to genetically resistant sheep.

A composite population of Haemonchus contortus was established with larvae from seven diverse sources, then maintained in Merino sheep bred to have either increased or decreased resistance to Haemonchus. After five, seven and 14 parasite generations, the two resulting lines of parasites were used to infect sheep from the increased resistance line, an unselected control line and the decreased resistance line. Line of sheep had a highly significant effect on average faecal egg counts 4 and 5 weeks after infection (geometric means 329, 735 and 1490 epg, respectively after the 14th generation), but the two lines of parasites yielded similar egg counts. There was no significant interaction between line of sheep and line of parasite, indicating that the parasite populations had not diverged significantly in their reproductive fitness, as measured by faecal egg count.     

Amphids: the neuronal ultrastructure of macrocyclic-lactone-resistant Haemonchus contortus

The development of anthelmintic resistance by nematode parasites is a growing problem for veterinarians and producers. The intensive use of the macrocyclic lactones for the treatment of a variety of parasitic diseases has hastened the development of resistance to this family of parasiticides among sheep, goats and cattle. As a result, resistance to ivermectin, moxidectin and doramectin by Haemonchus contortus has been documented throughout the world. While the exact sites of action of the macrocyclic lactones remain incompletely known, a critical point of entry for these drugs may be the terminally exposed sensory major neurons located in the cephalic end of the worms. These neurons, called amphidial neurons, are located in a pair of channels, the amphids, on either side of the pharynx and are exposed to the external environment via pores at the anterior tip of the worm. Through these neurons, important chemical and thermal cues are gathered by the parasite. Examination of serial electron micrographs of ivermectin-susceptible and ivermectin-resistant H. contortus allows for comparison of neuronal structure, arrangement of neurons within the amphidial channel, and distance of the tip of the dendritic processes to the amphidial pore. The latter of these characteristics provides a useful means by which to compare the association between the neurons and the external environment of the worm. Comparison of parental laboratory strains of ivermectin-susceptible H. contortus with related selected, ivermectin-resistant strains and with a wild-type ivermectin-susceptible field strain of H. contortus from Louisiana reveals that the ivermectin-resistant worms examined have markedly shorter sensory cilia than their ivermectin-susceptible parental counterparts. Additionally, the amphidial neurons of ivermectin-resistant worms are characterized by generalized degeneration and loss of detail, whereas other neurons outside of the channels, such as the labial and cephalic neurons, are normal in structure. Similar degeneration was also observed in field strains of doramectin-resistant H. contortus collected from a New Jersey alpaca heard. These findings raise a number of questions regarding the relationship between amphidial structure and macrocyclic lactone resistance as well as the role of amphids as a means of entry for these molecules. While shortened amphidial sensilla are associated with ivermectin resistance, it remains unclear if such a structural modification facilitates survival of nematodes exposed to the macrocyclic lactones.     

The mortality and fecundity of Haemonchus contortus in parasite-naive and parasite-exposed sheep following single experimental infections.

Parasite-exposed lambs and their parasite-naive controls were experimentally infected once only with 30,000 H. contortus larvae at 3, 9, 12, and 20 weeks following termination of a moderate immunizing infection of 30,000 H. contortus larvae. Previously exposed lambs, challenged at 3 weeks, had a significant reduction in the total H. contortus worm burden as compared to parasite-naive controls. No difference in the total H. contortus worm burden was found between parasite-exposed or parasite-naive lambs challenged at 9 weeks or thereafter. Female worms were found to be significantly smaller in lambs previously exposed to the parasite as compared to those found in parasite-naive lambs. The average parasite fecundity was 4700 eggs per female worm per day. Previous exposure of the lambs to the parasite had no effect on parasite fecundity. Various mathematical models were used to examine parasite fecundity. Parasite fecundity was found to increase in the initial post-challenge period reaching a constant value approximately 58 days after challenge infection. No density-dependent constraints on fecundity were observed.     

Genetics of mating and sex determination in the parasitic nematode Haemonchus contortus

Genetic analysis of parasitic nematodes has been a neglected area of research and the basic genetics of this important group of pathogens are poorly understood. Haemonchus contortus is one of the most economically significant livestock parasites worldwide and is a key experimental model for the strongylid nematode group that includes many important human and animal pathogens. We have undertaken a study of the genetics and the mode of mating of this parasite using microsatellite markers. Inheritance studies with autosomal markers demonstrated obligate dioecious sexual reproduction and polyandrous mating that are reported here for the first time in a parasitic helminth and provide the parasite with a mechanism of increasing genetic diversity. The karyotype of the H. contortus, MHco3(ISE) isolate was determined as 2n = 11 or 12. We have developed a panel of microsatellite markers that are tightly linked on the X chromosome and have used them to determine the sex chromosomal karyotype as XO male and XX female. Haplotype analysis using the X-chromosomal markers also demonstrated polyandry, independent of the autosomal marker analysis, and enabled a more direct estimate of the number of male parental genotypes contributing to each brood. This work provides a basis for future forward genetic analysis on H. contortus and related parasitic nematodes.     

Mucosal damage and neutropenia are required for Candida albicans dissemination

Candida albicans fungemia in cancer patients is thought to develop from initial gastrointestinal (GI) colonization with subsequent translocation into the bloodstream after administration of chemotherapy. It is unclear what components of the innate immune system are necessary for preventing C. albicans dissemination from the GI tract, but we have hypothesized that both neutropenia and GI mucosal damage are critical for allowing widespread invasive C. albicans disease. We investigated these parameters in a mouse model of C. albicans GI colonization that led to systemic spread after administration of immunosuppression and mucosal damage. After depleting resident GI intestinal flora with antibiotic treatment and achieving stable GI colonization levels of C. albicans, it was determined that systemic chemotherapy with cyclophosphamide led to 100% mortality, whereas selective neutrophil depletion, macrophage depletion, lymphopenia or GI mucosal disruption alone resulted in no mortality. Selective neutrophil depletion combined with GI mucosal disruption led to disseminated fungal infection and 100% mortality ensued. GI translocation and dissemination by C. albicans was also dependent on the organism's ability to transform from the yeast to the hyphal form. This mouse model of GI colonization and fungemia is useful for studying factors of innate host immunity needed to prevent invasive C. albicans disease as well as identifying virulence factors that are necessary for fungal GI colonization and dissemination. The model may also prove valuable for evaluating therapies to control C. albicans infections.     

The murine model of infection with Leishmania major and its importance for the deciphering of mechanisms underlying differences in Th cell differentiation in mice from different genetic backgrounds

Mice from the majority of inbred strains are resistant to infection by Leishmania major, an obligate intracellular protozoan parasite of macrophages in the mammalian host. In contrast, mice from BALB strains are unable to control infection and develop progressive disease. In this model of infection, genetically determined resistance and susceptibility have been clearly shown to result from the appearance of parasite-specific CD4+ T helper 1 or T helper 2 cells, respectively. This murine model of infection is considered as one of the best experimental systems for the study of the mechanisms operating in vivo at the initiation of polarised T helper 1 and T helper 2 cell maturation. Among the several factors influencing Th cell development, cytokines themselves critically regulate this process. The results accumulated during the last years have clarified some aspects of the role played by cytokines in Th cell differentiation. They are providing critical information that may ultimately lead to the rational devise of means by which to tailor immune responses to the effector functions that are most efficient in preventing and/or controlling infections with pathogens.     

Seasonal Patterns of Hormones,Macroparasites, and Microparasites in Wild African Ungulates: The Interplay among Stress,Reproduction, and Disease

Sex hormones, reproductive status, and pathogen load all affect stress. Together with stress, these factors can modulate the immune system and affect disease incidence. Thus, it is important to concurrently measure these factors, along with their seasonal fluctuations, to better understand their complex interactions. Using steroid hormone metabolites from fecal samples, we examined seasonal correlations among zebra and springbok stress, reproduction, gastrointestinal (GI) parasite infections, and anthrax infection signatures in zebra and springbok in Etosha National Park (ENP), Namibia, and found strong seasonal effects. Infection intensities of all three GI macroparasites examined (strongyle helminths, Strongyloides helminths, and Eimeria coccidia) were highest in the wet season, concurrent with the timing of anthrax outbreaks. Parasites also declined with increased acquired immune responses. We found hormonal evidence that both mares and ewes are overwhelmingly seasonal breeders in ENP, and that reproductive hormones are correlated with immunosuppression and higher susceptibility to GI parasite infections. Stress hormones largely peak in the dry season, particularly in zebra, when parasite infection intensities are lowest, and are most strongly correlated with host mid-gestation rather than with parasite infection intensity. Given the evidence that GI parasites can cause host pathology, immunomodulation, and immunosuppression, their persistence in ENP hosts without inducing chronic stress responses supports the hypothesis that hosts are tolerant of their parasites. Such tolerance would help to explain the ubiquity of these organisms in ENP herbivores, even in the face of their potential immunomodulatory trade-offs with anti-anthrax immunity.     

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.