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.     

Global patterns reveal strong population structure in Haemonchus contortus, a nematode parasite of domesticated ruminants

We have examined the global population genetic structure of Haemonchus contortus. The genetic variability was studied using both amplified fragment length polymorphism (AFLP) and nad4 sequences of the mitochondrial genome. To examine the performance and information content of the two different marker systems, comparative assessment of population genetic diversity was undertaken in 19 isolates of H. contortus, a parasitic nematode of small ruminants. A total of 150 individual adult worms representing 14 countries from all inhabited continents were analysed. Altogether 1,429 informative AFLP markers were generated using four different primer combinations. Also, the genetic variation was high, which agrees with results from previous AFLP studies of nematode parasites of livestock. The genetic structure was high, indicating limited gene flow between the different isolates and populations from each continent mostly formed monophyletic groups in the phylogenetic analysis. However, for isolates representing Australia, Greece and one laboratory strain that originated from South Africa (WRS), there was no clear genetic relationship between the isolates and the distance between their geographical origins. Basically the same pattern was observed for the mitochondrial marker, although the phylogenetic analysis was less resolved than for AFLP. In contrast with previous findings on the population genetic structure of H. contortus, the calculation of population structure gave high values (Nst=0.59). The strong structure was present also for the four Swedish isolates (Nst=0.16) representing a small geographical area.     

Ivermectin resistant and susceptible third-stage larvae of Haemonchus contortus: cholinesterase and phosphatase activities

Cholinesterase and acid phosphatase (AP), but not alkaline phosphatase activities, were detected in cytosolic and membrane-bound fractions of ivermectin resistant and susceptible Haemonchus contortus infective-stage larvae. Some differences in acetylcholinesterase activity of cytosolic fractions and in the AP activity of these fractions as well as in the response to AP inhibitors by membrane-bound fractions were detected. Data are discussed.     

New oral linguiform projections and their associated neurons in the third-stage infective larva of the parasitic nematode Oesophagostomum dentatum

The infective larvae (L3i) of the nematode parasite of swine, Oesophagostomum dentatum, are passively ingested by their hosts. The L3i exhibit certain behaviors that are probably selected to increase the likelihood of ingestion, by strategic positioning in the environment. The larvae show positive geotactic behavior and respond to temperature variations in their environment, as shown by their behavior on a thermal gradient. To investigate neuronal control of this behavior, we initiated a study of the structure of the amphidial neurons of this parasite. The same number and types of neuronal dendritic processes are found in the amphids of the O. dentatum L3i as in those of its close relatives Haemonchus contortus and Ancylostoma caninum. Well-developed dendritic processes of wing cells are located in the amphidial sheath cells, these being similar to wing cells AWA in the free-living nematode Caenorhabditis elegans but actually more extensive. Similar to its close relatives just mentioned, and C. elegans as well, O. dentatum L3i has prominent finger cell processes, the finger cell neurons being the thermoreceptors in all 3 of the preceding species. However, unlike the arrangement seen in H. contortus and A. caninum, where the microvilli-like "fingers" of these neurons lie dorsal to the amphidial channel and occupy a very large portion (>50%) of the anterior end of the larva, the dendritic process of the finger cells in O. dentatum extends into unusual linguiform projections that, in turn, extend into the lumen of the mouth tube, a complex structural arrangement that has not been described for any other nematode.     

From isolates to a synthetic laboratory population: maintenance of variability in the nematode Haemonchus contortus

Haemonchus contortus isolates were collected from goats of five locations with different climatic characteristics in Guadeloupe archipelago. They were investigated for morphology, morphometrics and allozyme diversity after passage in immunosuppressed lambs using long acting corticoids. The basic aim of the work was to construct a synthetic strain in laboratory conditions which was representative of the isolates. The isolates were only slightly different although climatic conditions were very different. The resemblance of isolates might be due to the practice of goat exchanges between farms or to their insular origin. However the isolate from a smaller island (Les Saintes) was different (mostly on morphometrics) from all the others originating from Guadeloupe main Island. The first assemblage in laboratory resulting from the installation from a mixture of the five isolates was not very representative, whereas the next generation (synthetic strain) resembled all the isolates as shown from allozyme study. Female fecundity and length in established synthetic strain were lower than that recorded in isolates, indicating a decrease in fitness, possibly due to the stability of experimental environment. The representativity of the synthetic strain was good but the strain could still evaluate on further passages and should be evaluated on a large number of generations maintained in laboratory.     

The development of vaccines against gastrointestinal nematode parasites, particularly Haemonchus contortus.

Many parasitic nematodes are developing resistance to chemical treatment, and the search is on to produce commercially viable molecular vaccines. Much progress has been made with highly protective 'hidden antigens', especially for Haemonchus contortus, and recent work with new 'natural antigens' has yielded promising results. Here, Sue Newton and Ed Munn review the most recent advances in these two main approaches to this problem.     

A unique thioredoxin of the parasitic nematode Haemonchus contortus with glutaredoxin activity

The dependency of parasites on the cellular redox systems has led to their investigation as novel drug targets. Defence against oxidative damage is through the thioredoxin and glutathione systems. The classic thioredoxin is identified by the active site Cys-Gly-Pro-Cys (CGPC). Here we describe the identification of a unique thioredoxin in the parasitic nematode, Haemonchus contortus. This thioredoxin-related protein, termed HcTrx5, has an arginine in its active site (Cys-Arg-Ser-Cys; CRSC) that is not found in any other organism. Recombinant HcTrx5 was able to reduce the disulfide bond in insulin, and be regenerated by mammalian thioredoxin reductase with a K_m 2.19 ± 1.5 μM, similar to the classic thioredoxins. However, it was also able to reduce insulin when glutathione and glutathione reductase replaced the thioredoxin reductase. When coupled with H. contortus peroxiredoxin, HcTrx5 was active using either the thioredoxin reductase or the glutathione and glutathione reductase. HcTrx5 is expressed through the life cycle, with highest expression in the adult stage. The unique activity of this thioredoxin makes it a potential drug target for the control of this parasite.     

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.     

A preliminary study of the effect of microclimate on third-stage larvae of Haemonchus contortus and Haemonchus placei on irrigated pasture.

Assessments were made on the influence of several microclimatic variables on the availability of third-stage larvae of Haemonchus contortus and Haemonchus placei on four strata of irrigated Kikuyu pasture. Three replicates of these pasture samples were collected on 18 sample days over 12 months and the log10 mean counts of the larvae recovered were analysed by a step-wise regression model. Predictors for the log counts of the four strata for the two nematode species included relative humidity, illumination, air temperature and windspeed. The effect of air temperature on larvae of both Haemonchus species was similar; as air temperature increased, the number of larvae on pasture increased. The inverse was true for windspeed; as windspeed increased larval counts decreased. For H. contortus, relative humidity increased as the number of larvae increased on all strata except upper herbage. The R2 values ranged from 0.11 to 0.21 for H. contortus and from 0.04 to 0.12 for H. placei. Under the conditions of this study, only 21% of the effect on H. contortus and 12% on H. placei third-stage larvae on pasture can be explained by microclimatic conditions.     

Genetic analysis of inbreeding of two strains of the parasitic nematode Haemonchus contortus

Haemonchus contortus is a sheep parasitic nematode that causes severe economic losses. Previous studies have indicated a high degree of genetic heterogeneity, which is hardly affected by selection for drug resistance. As a tool for the analysis of the population dynamics of H. contortus and its response to drug resistance, we designed a strategy to study the inbreeding of a benzimidazole-sensitive and a benzimidazole-resistant strain. After 15 generations, a theoretical inbreeding coefficient of 0.87 was achieved. The different stages of inbreeding were analysed using restriction fragment polymorphism, microsatellite variability and amplified fragment length polymorphism. Model-based clustering of the amplified fragment length polymorphism genotypes showed that the allele frequencies of the benzimidazole-resistant strain were stable during the last eight generations. In the sensitive strain a gradual shift of allele frequencies was observed, which led to a temporary increase of the genetic diversity around the eight generations.     

Thioredoxins of a parasitic nematode: comparison of the 16- and 12-kDA thioredoxins from Haemonchus contortus

Thioredoxins are a family of small proteins conserved through evolution, which are essential for the maintenance of cellular homeostasis. The "classic" thioredoxin, identified in most species, is a 12-kDa protein with a Cys-Pro-Gly-Cys (CPGC) active site. However, in nematodes a larger protein, 16 kDa, with a Cys-Pro-Pro-Cys (CPPC) active site was identified. We report that in the parasitic nematode Haemonchus contortus, both the 12-kDa (HcTrx1) and the 16-kDa (HcTrx3) species are expressed through the life cycle. However, the HcTrx3 is expressed at higher concentrations. Recombinant HcTrx1 and HcTrx3 were produced and both reduced insulin at a rate similar to that observed with ovine (host) and Escherichia coli thioredoxins and both were regenerated by a mammalian thioredoxin reductase, demonstrating that they have similar thioredoxin activity. Unlike mammalian thioredoxins, both proteins were able to reduce oxidised glutathione and hydrogen peroxide. This suggests essential roles for these proteins in response to oxidative stress and the host immune attack. Analysis of ivermectin-resistant H. contortus showed that expression of both genes were increased in a drug-resistant strain relative to a sensitive strain. Involvement in drug resistance identifies these thioredoxin proteins as potential drug targets for parasite control.     

Arginine metabolism in the sheep abomasal nematode parasites Haemonchus contortus and Teladorsagia circumcincta

The ornithine urea cycle, polyamine synthesis, nitric oxide synthesis and metabolism of arginine to putrescine have been investigated in L3 and adult Haemonchus contortus and Teladorsagia circumcincta. Neither parasite had a detectable arginine deiminase/dihydrolase pathway nor a functional ornithine urea cycle. Nitric oxide synthase was present in central and peripheral nerves, but was not detected in whole parasite homogenates. Both arginase (E.C. 3.5.3.1) and agmatinase (E.C. 3.5.3.11) activities were present in both species. Arginase did not require added Mn²⁺ and had an optimal pH of 8.5. Polyamine metabolism differed in the two species and from that in mammals. Ornithine decarboxylase (E.C. 4.1.1.17) was present in both parasites, but no arginine decarboxylase (E.C. 4.1.1.19) activity was detected in T. circumcincta. The flexibility of synthesis of putrescine in H. contortus may make this pathway less useful as a target for parasite control than in T. circumcincta, in which only the ornithine decarboxylase pathway was detected.