Peter W. Hunt

A hemoglobin from plants homologous to truncated hemoglobins of microorganisms

We have identified a nuclear-encoded Hb from plants (GLB3) that has a central domain similar to the “truncated” Hbs of bacteria, protozoa, and algae. The three-dimensional structure of these Hbs is a 2-on-2 arrangement of α-helices, distinct from the 3-on-3 arrangement of the standard globin fold [Pesce, A., Couture, M., Dewilde, S., Guertin, M., Yamauchi, K., Ascenzi, P., Moens, L. & Bolognesi, M. (2000) EMBO J. 19, 2424–2434]. GLB3-like genes are not found in animals or yeast, but our analysis reveals that they are present in a wide range of Angiosperms and a Bryophyte. Although cyanobacteria and Chlamydomonas have 2-on-2 Hbs (GLBN), GLB3 is more likely related to GLBO-type 2-on-2 Hbs from bacteria. Consequently, GLB3 is unlikely to have arisen from a horizontal transfer between the chloroplast and nuclear genomes. Arabidopsis thaliana GLB3 protein exhibits unusual concentration-independent binding of O2 and CO. The absorbance spectrum of deoxy-GLB3 is unique; the protein forms a transient six-coordinate structure after reduction and deoxygenation, which slowly converts to a five-coordinate structure. In A. thaliana, GLB3 is expressed throughout the plant but responds to none of the treatments that induce plant 3-on-3 Hbs. Our analysis of the sequence, ligand interactions, and expression profile of GLB3 indicates that this protein has unique biochemical properties, evolutionary history, and, most likely, a function distinct from those of other plant Hbs.

Increased level of hemoglobin 1 enhances survival of hypoxic stress and promotes early growth in Arabidopsis thaliana

Overexpression of a class 1 Hb (GLB1) protects Arabidopsis thaliana plants from the effects of severe hypoxia. Overexpression of the bifunctional symbiotic Hb (GLB1S) from Parasponia andersonii in A. thaliana also increases survival after hypoxia. Plants overexpressing the Hb 1 protein, mutated to have a low oxygen affinity, are as susceptible to hypoxia as WT plants, suggesting that the protection against hypoxia depends on the ability of the Hb to bind ligands, such as oxygen, with high affinity. A mild hypoxia pretreatment (5%) induces the Hb gene and increases the survival of plants after severe hypoxic treatment (0.1%). These results with Hb 1 show that plant Hbs have a role other than in nitrogen-fixing root nodules. Plants overexpressing the GLB1 protein show early vigorous growth in nonhypoxic conditions and are 50% larger in weight than the controls at 14 days. The constitutive expression of GLB1 also resulted in a reduced number of root hairs and increased number of laterals in the root system.

Toward practical, DNA-based diagnostic methods for parasitic nematodes of livestock -Bionomic and biotechnological implications

Parasitic nematodes of livestock have a major economic impact worldwide. In spite of the health problems caused by nematodes and advances toward the development of vaccines and new therapeutic agents against some of them, relatively limited attention has been paid to the need for improved, practical methods of diagnosis. Accurate diagnosis and genetic characterization of parasitic nematodes of livestock are central to their effective control, particularly given the current, serious problems with anthelmintic resistance in nematode populations. Traditional diagnostic techniques have considerable limitations, and there have been some advances toward the development of molecular-diagnostic tools. This article provides a brief account of the significance of parasitic nematodes (order Strongylida), reviews the techniques that have been evaluated or used for diagnosis and describes developments in polymerase chain reaction (PCR)-based methods for the specific diagnosis of nematode infection/s and the genetic characterisation of the causative agents. The advances made in recent years provide a solid foundation for the development of practical, highly sensitive and specific diagnostic tools for epidemiological investigations and for use in control programmes.

Mapping SNP-anchored genes using high-resolution melting analysis in almond

Peach and almond have been considered as model species for the family Rosaceae and other woody plants. Consequently, mapping and characterisation of genes in these species has important implications. High-resolution melting (HRM) analysis is a recent development in the detection of SNPs and other markers, and proved to be an efficient and cost-effective approach. In this study, we aimed to map genes corresponding to known proteins in other species using the HRM approach. Prunus unigenes were searched and compared with known proteins in the public databases. We developed single-nucleotide polymorphism (SNP) markers, polymorphic in a mapping population produced from a cross between the cloned cultivars Nonpareil and Lauranne. A total of 12 SNP-anchored putative genes were genotyped in the population using HRM, and mapped to an existing linkage map. These genes were mapped on six linkage groups, and the predicted proteins were compared to putative orthologs in other species. Amongst those genes, four were abiotic stress-responsive genes, which can provide a starting point for construction of an abiotic resistance map. Two allergy and detoxification related genes, respectively, were also mapped and analysed. Most of the investigated genes had high similarities to sequences from closely related species such as apricot, apple and other eudicots, and these are putatively orthologous. In addition, it was shown that HRM can be an effective means of genotyping populations for the purpose of constructing a linkage map. Our work provides basic genomic information for the 12 genes, which can be used for further genetic and functional studies.

Relative level of thiabendazole resistance associated with the E198A and F200Y SNPs in larvae of a multi-drug resistant isolate of Haemonchus contortus.

While the F200Y SNP in the beta-tubulin gene is most commonly associated with benzimidazole resistance in trichostrongylid nematodes, other SNPs as well as drug efflux pathways have been implicated in the resistance. The relative contributions of all these mechanisms are not understood sufficiently to allow expected drug efficacy to be inferred from molecular data. As a component of developing better means to interpret molecular resistance tests, the present study utilised a drug resistant Haemonchus contortus isolate which possesses two of the principal benzimidazole resistance SNPs (E198A and F200Y) in order to assess the relative degree of resistance conferred by the two SNPs. We exposed larvae to a range of thiabendazole concentrations in in vitro development assays, and collected the surviving L3 larvae at each drug concentration to establish sub-populations showing increasing levels of resistance. We then sequenced the isotype 1 beta-tubulin gene in pooled larval samples, and measured allele frequencies at the two SNP positions. The frequency of the resistance allele at the 198 position increased as the thiabendazole concentration increased, while the frequency of the resistance allele at the 200 position decreased. Genotyping of individual larvae showed that the highest drug concentration was associated with the removal of all genotypes except for homozygous resistance at the 198 position alongside homozygous susceptible at the 200 position. This indicates that, at least for larval life stages, the E198A SNP is able to confer higher levels of resistance to benzimidazole drugs than the F200Y SNP, and that the homozygosity at 198 in the highly resistant individuals is mutually exclusive with heterozygosity or resistant homozygosity at the 200 position. This study illustrates the need to understand the relative contributions of different resistance mechanisms in order to maximise the degree to which molecular tests are able to inform on drug resistance phenotype.

Molecular diagnosis of infections and resistance in veterinary and human parasites

Since 1977, >2000 research papers described attempts to detect, identify and/or quantify parasites, or disease organisms carried by ecto-parasites, using DNA-based tests and 148 reviews of the topic were published. Despite this, only a few DNA-based tests for parasitic diseases are routinely available, and most of these are optional tests used occasionally in disease diagnosis. Malaria, trypanosomiasis, toxoplasmosis, leishmaniasis and cryptosporidiosis diagnosis may be assisted by DNA-based testing in some countries, but there are very few cases where the detection of veterinary parasites is assisted by DNA-based tests. The diagnoses of some bacterial (e.g. lyme disease) and viral diseases (e.g. tick borne encephalitis) which are transmitted by ecto-parasites more commonly use DNA-based tests, and research developing tests for these species makes up almost 20% of the literature. Other important uses of DNA-based tests are for epidemiological and risk assessment, quality control for food and water, forensic diagnosis and in parasite biology research. Some DNA-based tests for water-borne parasites, including Cryptosporidium and Giardia, are used in routine checks of water treatment, but forensic and food-testing applications have not been adopted in routine practice. Biological research, including epidemiological research, makes the widest use of DNA-based diagnostics, delivering enhanced understanding of parasites and guidelines for managing parasitic diseases. Despite the limited uptake of DNA-based tests to date, there is little doubt that they offer great potential to not only detect, identify and quantify parasites, but also to provide further information important for the implementation of parasite control strategies. For example, variant sequences within species of parasites and other organisms can be differentiated by tests in a manner similar to genetic testing in medicine or livestock breeding. If an association between DNA sequence and phenotype has been demonstrated, then qualities such as drug resistance, strain divergence, virulence, and origin of isolates could be inferred by DNA-based tests. No such tests are in clinical or commercial use in parasitology and few tests are available for other organisms. Why have DNA-based tests not had a bigger impact in veterinary and human medicine? To explore this question, technological, biological, economic and sociological factors must be considered. Additionally, a realistic expectation of research progress is needed. DNA-based tests could enhance parasite management in many ways, but patience, persistence and dedication will be needed to achieve this goal.

DNA-based methodology for the quantification of gastrointestinal nematode eggs in sheep faeces

The presence of gastrointestinal nematode eggs in faecal samples is diagnostic of infection by these parasites. However, this technique cannot be used to distinguish between species of importance. The faecal culture technique and subsequent microscopic analysis of developed larvae is currently used to determine which parasite species are present in the samples, but these techniques take a week to perform and have inherent limitations. To overcome these parasite detection and identification problems, we have developed a DNA extraction method for sheep faeces, and a quantitative multiplex PCR (qPCR) test which can both enumerate and identify Haemonchus, Trichostrongylus and Teladorsagia. We demonstrate that the technique is sensitive to 10 eggs per gram and that dilution of DNA to 0.1 fold can overcome PCR inhibition issues for samples obtained from the field, while maintaining assay sensitivity. Further development of these tests for commercial use is warranted, given their potential to provide consistently faster and more accurate diagnoses of these parasites using simple sample collection and laboratory methods which can be easily adapted for the detection of a variety of pathogens from the same faecal sample.

Development of a modified molecular diagnostic procedure for the identification and quantification of naturally occurring strongylid larvae on pastures

A molecular procedure was developed to detect and quantify larvae of different strongylid parasite species recovered from pasture samples. Two lamb flocks (L and S) grazed separate paddocks with different natural larvae challenges (one low [Paddock L] and one high [Paddock S] challenge) on a commercial farm in Western Australia. Pasture samples were collected and analysed for larvae on 9 separate occasions from each paddock. Pregnant Merino ewes were sampled on 3 separate occasions (2 pre-partum and 1 post-partum). Following lambing, 203 female crossbred lambs were identified, from which faecal samples were collected across five separate samplings. Lamb production and faecal attributes were recorded. Genomic DNA was extracted directly from lamb faeces, in addition to the genomic DNA extracts from strongylid larval species recovered from pastures. Faecal worm egg counts (FWECs) were undertaken. Species-specific qPCRs and conventional PCRs (ITS-2 nuclear ribosomal DNA) were used to screen samples for strongylid species (Teladorsagia circumcincta, Trichostrongylus spp., Haemonchus contortus, Chabertia ovina and Oesophagostomum venulosum). Negative correlations (r(2)>0.91) were found between qPCR C(q) values and log-transformed pasture larval counts for Trichostrongylus spp. and T. circumcincta. Moderate levels of agreement between pasture larval counts and qPCR results were observed (67%). A clear difference in pasture larval challenge levels was observed between the two flocks using both qPCR and conventional pasture larval counts. It is difficult to draw conclusions on the production performances of lambs from the two experimental flocks, as no further replicates were able to be conducted following this experiment. Flock L had higher dressing percentages than Flock S (P=0.038), along with significantly higher faecal consistency and breech fleece faecal soiling scores at successive samplings. The molecular procedures utilised in this study have the potential to be beneficial for livestock grazing management strategies and parasite surveillance, however further investigation is necessary before they can become part of routine diagnostics.

Trichostrongylus colubriformis larvae induce necrosis and release of IL33 from intestinal epithelial cells in vitro: implications for gastrointestinal nematode vaccine design.

Gastrointestinal nematodes represent a major production problem for ruminant livestock. Enhancing immunity to gastrointestinal nematodes through vaccination is desirable but mechanistic understanding of initial host responses that facilitate gastrointestinal nematode protective immunity is limited. We hypothesise that gastrointestinal nematode invasion induces mucosal epithelium damage and alarmin (e.g. IL33) release, thereby contributing to initiation of protective gastrointestinal nematode immunity. To test this, an in vitro air-liquid interface human HT-29 epithelial cell-Trichostrongylus colubriformis co-culture system was developed. Exsheathed L3 T. colubriformis exhibited both sinusoidal and burrowing motions in the co-culture system. Burrowing parasites, but not ivermectin-paralysed larvae, induced necrotic death of epithelial cells (annexin V(+)/propidium iodide(+)/caspase 3/7(-)). Microscopy confirmed that larvae consumed labelled necrotic epithelial cell contents. Trichostrongylus colubriformis larvae and their post-exsheathment antigens (excretory/secretory products) significantly induced IL33 mRNA expression in the epithelial cells. Immunoblot confirmed that IL33 was released from epithelial cells due to the damage caused by motile larvae. Exposure of HT-29 cells to alum or Sigma proprietary adjuvants induced significant epithelial cell IL33 mRNA expression without inducing cellular necrosis. Hence, the intracellular contents were not released externally where they might exert alarmin activity and this may limit their ability to trigger a protective anti-gastrointestinal nematode response. We conclude that T. colubriformis motion at the infection site induces intestinal epithelial cell necrosis which facilitates the release of intracellular contents, including IL33, and may be fundamental to the initiation of an appropriate host response to gastrointestinal nematodes. Our co-culture model is useful for studying initial epithelial cell-parasite interactions without conducting expensive animal trials.

Larval development assays reveal the presence of sub-populations showing high- and low-level resistance in a monepantel (Zolvix®)-resistant isolate of Haemonchus contortus

Resistance to the amino-acetonitrile derivative monepantel has been reported in several species of gastrointestinal nematodes over recent years. We were interested in the use of in vitro assays with free-living worm life-stages to detect resistance to this drug. We therefore used larval development and larval migration assays to examine dose response relationships for the drug against two susceptible and one resistant isolate of Haemonchus contortus. The resistant isolate was established by laboratory propagation of the survivors of a field treatment with Zolvix(®) that had originally resulted in a drug efficacy of over 99%. Drug efficacy against this field-derived laboratory-propagated resistant isolate in vivo was approximately 15%. The larval development assay proved able to discriminate between the susceptible and resistant isolates, with larvae of the resistant isolate showing an ability to develop at higher drug concentrations than the two susceptible isolates. The resistant isolate showed the presence of two distinct subpopulations, separated by a plateau in the dose-response curve. Sub-population 1 (approximately 40% of the total population) showed a low level of resistance with an IC50 increased approximately 7-fold compared to the baseline susceptible isolate, while sub-population 2 (the remaining 60% of the total population) showed an IC50 increased over 1000-fold compared to the baseline susceptible isolate. This level of resistance is unusually high for any gastrointestinal nematode species in drug dose-response in vitro assays. In contrast, the migration assay could not discriminate between the three isolates, with migration not reduced to zero at any of the drug concentrations tested. This study demonstrates that a larval development assay is able to detect resistance to monepantel in H. contortus, and that resistance can exist in two distinct forms. This suggests that at least two separate monepantel resistance mechanisms are acting within the worm isolate studied here, with one or more mechanisms conferring a much higher level of resistance than the other(s).