DNA technological progress toward advanced diagnostic tools to support human hookworm control

Blood-feeding hookworms are parasitic nematodes of major human health importance. Currently, it is estimated that 740 million people are infected worldwide, and more than 80 million of them are severely affected clinically by hookworm disease. In spite of the health problems caused and the advances toward the development of vaccines against some hookworms, limited attention has been paid to the need for improved, practical methods of diagnosis. Accurate diagnosis and genetic characterization of hookworms is central to their effective control. While traditional diagnostic methods have considerable limitations, there has been some progress toward the development of molecular-diagnostic tools. The present article provides a brief background on hookworm disease of humans, reviews the main methods that have been used for diagnosis and describes progress in establishing polymerase chain reaction (PCR)-based methods for the specific diagnosis of hookworm infection and the genetic characterisation of the causative agents. This progress provides a foundation for the rapid development of practical, highly sensitive and specific diagnostic and analytical tools to be used in improved hookworm prevention and control programmes.     

Advances in the diagnosis of key gastrointestinal nematode infections of livestock,with an emphasis on small ruminants

Parasitic nematodes (roundworms) of livestock have major economic impact globally. In spite of the diseases caused by these nematodes and some advances in the design of new therapeutic agents (anthelmintics) and attempts to develop vaccines against some of them, there has been limited progress in the establishment of practical diagnostic techniques. The specific and sensitive diagnosis of gastrointestinal nematode infections of livestock underpins effective disease control, which is highly relevant now that anthelmintic resistance (AR) is a major problem. Traditional diagnostic techniques have major constraints, in terms of sensitivity and specificity. The purpose of this article is to provide a brief background on gastrointestinal nematodes (Strongylida) of livestock and their control; to summarize conventional methods used for the diagnosis and discuss their constraints; to review key molecular-diagnostic methods and recent progress in the development of advanced amplification-based and sequencing technologies, and their implications for epidemiological investigations and the control of parasitic diseases.     

Cryptosporidium--biotechnological advances in the detection, diagnosis and analysis of genetic variation

Cryptosporidiosis is predominantly a gastrointestinal disease of humans and other animals, caused by various species of protozoan parasites representing the genus Cryptosporidium. This disease, transmitted mainly via the faecal-oral route (in water or food), is of major socioeconomic importance worldwide. The diagnosis and genetic characterization of the different species and population variants (usually recognised as "genotypes" or "subgenotypes") of Cryptosporidium is central to the prevention, surveillance and control of cryptosporidiosis, particularly given that there is presently no broadly applicable treatment regimen for this disease. Although traditional phenotypic techniques have had major limitations in the specific diagnosis of cryptosporidiosis, there have been major advances in the development of molecular analytical and diagnostic tools. This article provides a concise account of Cryptosporidium and cryptosporidiosis, and focuses mainly on recent advances in nucleic acid-based approaches for the diagnosis of cryptosporidiosis and analysis of genetic variation within and among species of Cryptosporidium. These advances represent a significant step toward an improved understanding of the epidemiology as well as the prevention and control of cryptosporidiosis.     

Biotechnological advances in the diagnosis of avian coccidiosis and the analysis of genetic variation in Eimeria

Coccidiosis is an intestinal disease of chickens caused by various species of protozoan parasites within the genus Eimeria. This disease has a major economic impact to growers and to the poultry industry world-wide. The diagnosis and genetic characterization of the different species of Eimeria are central to the prevention, surveillance and control of coccidiosis, particularly now given the major problems with wide-spread resistance of Eimeria species against anticoccidial drugs (coccidiostats) and the residue problems associated with these compounds. While traditional methods have had major limitations in the specific diagnosis of coccidiosis, there have been significant advances in the development of molecular-diagnostic tools. The present article provides a background on coccidiosis, reviews the main molecular methods which have been used and describes recent advances in the establishment of polymerase chain reaction (PCR)-coupled electrophoretic approaches for the specific diagnosis of coccidiosis as well as the genetic characterization of species of Eimeria. These biotechnological advances are considered to represent a significant step toward the improved prevention and control of this important disease of poultry.     

Biotechnological advances in the diagnosis of avian coccidiosis and the analysis of genetic variation in Eimeria

Coccidiosis is an intestinal disease of chickens caused by various species of protozoan parasites within the genus Eimeria. This disease has a major economic impact to growers and to the poultry industry world-wide. The diagnosis and genetic characterization of the different species of Eimeria are central to the prevention, surveillance and control of coccidiosis, particularly now given the major problems with wide-spread resistance of Eimeria species against anticoccidial drugs (coccidiostats) and the residue problems associated with these compounds. While traditional methods have had major limitations in the specific diagnosis of coccidiosis, there have been significant advances in the development of molecular-diagnostic tools. The present article provides a background on coccidiosis, reviews the main molecular methods which have been used and describes recent advances in the establishment of polymerase chain reaction (PCR)-coupled electrophoretic approaches for the specific diagnosis of coccidiosis as well as the genetic characterization of species of Eimeria. These biotechnological advances are considered to represent a significant step toward the improved prevention and control of this important disease of poultry.     

Molecular approaches for studying ascaridoid nematodes with zoonotic potential, with an emphasis on Toxocara species

Species-specific identification of ascaridoid nematodes at any developmental stage is a prerequisite for detailed investigation of the life cycles, systematics and epidemiology of this important group, and is also crucial for the diagnosis of associated infections. The morphological identification of some species and/or their larval stages can, however, present considerable difficulty. Recently, PCR-based methods, using genetic markers in the internal transcribed spacers (ITS) of ribosomal DNA, have been shown to provide reliable alternatives to more traditional methods for the specific identification of nematodes. This article provides an account of recent research on the development of PCR-based methods (utilizing ITS sequences) for the specific identification of ascaridoid nematodes of zoonotic potential, for the diagnosis of infections, and for the analysis of genetic variation within and among individual nematodes and their populations. Prospects for using these diagnostic and analytical tools to investigate epidemiological and population genetic questions relating to ascaridoid parasites are also discussed.     

Toward next-generation sequencing of mitochondrial genomes — Focus on parasitic worms of animals and biotechnological implications

Helminths (worms) include parasitic nematodes (roundworms) and platyhelminths (flatworms). These worms are abundant, and many of them are of agricultural, aquacultural, veterinary and medical importance and cause substantial socioeconomic losses worldwide. The genetic characterization of parasitic nematodes using advanced molecular tools is central to the diagnosis of infections and the control of parasitism. The accurate analysis of genetic variation also underpins studies of their taxonomy, epidemiology and evolutionary history. Although the nuclear genome contains suitable genetic markers (e.g., in ribosomal DNA) for the identification of many species, the large size and high variability of the mt genome consistently provides a rich source of such markers for informative systematic and epidemiological studies both within and among species. There is significant value in establishing a practical platform for the rapid sequencing, annotation and analysis of mt genomic datasets to underpin such fundamental and applied studies of parasitic worms (= helminths). In the last decade, there have been some important advances in the mt genomics of helminths, but next-generation sequencing (NGS) technologies now provide opportunities for high throughput sequencing, assembly and annotation. In this article, we provide a background on mt genomics, cover technological challenges and recent advances, and provide a perspective on future mt genome research of parasitic helminths and its fundamental scientific and biotechnological implications.     

Rendering the Intractable More Tractable: Tools from Caenorhabditis elegans Ripe for Import into Parasitic Nematodes

Recent and rapid advances in genetic and molecular tools have brought spectacular tractability to Caenorhabditis elegans, a model that was initially prized because of its simple design and ease of imaging. C. elegans has long been a powerful model in biomedical research, and tools such as RNAi and the CRISPR/Cas9 system allow facile knockdown of genes and genome editing, respectively. These developments have created an additional opportunity to tackle one of the most debilitating burdens on global health and food security: parasitic nematodes. I review how development of nonparasitic nematodes as genetic models informs efforts to import tools into parasitic nematodes. Current tools in three commonly studied parasites (Strongyloides spp., Brugia malayi, and Ascaris suum) are described, as are tools from C. elegans that are ripe for adaptation and the benefits and barriers to doing so. These tools will enable dissection of a huge array of questions that have been all but completely impenetrable to date, allowing investigation into host–parasite and parasite–vector interactions, and the genetic basis of parasitism.     

RNAi in Haemonchus contortus: a potential method for target validation

RNA interference (RNAi) is a method for the functional analysis of specific genes, and is particularly well developed in the free-living nematode Caenorhabditis elegans. There have been several attempts to apply this method to parasitic nematodes. In a recent study undertaken in Haemonchus contortus, Geldhof and colleagues concluded that, although a mechanism for RNAi existed, the methods developed for RNAi in C. elegans had variable efficacy in this parasitic nematode. The potential benefits of RNAi are clear; however, further studies are required to characterize the mechanism present in parasitic nematodes, and to improve culture systems for these nematodes to monitor the long-term effects of RNAi. Only then could RNAi become a reliable assay of gene function.     

Nematode cys-loop GABA receptors: biological function, pharmacology and sites of action for anthelmintics

Parasitic nematode infection of humans and livestock is a major problem globally. Attempts to control nematode populations have led to the development of several classes of anthelmintic, which target cys-loop ligand-gated ion channels. Unlike the vertebrate nervous system, the nematode nervous system possesses a large and diversified array of ligand-gated chloride channels that comprise key components of the inhibitory neurotransmission system. In particular, cys-loop GABA receptors have evolved to play many fundamental roles in nematode behaviour such as locomotion. Analysis of the genomes of several free-living and parasitic nematodes suggests that there are several groups of cys-loop GABA receptor subunits that, for the most part, are conserved among nematodes. Despite many similarities with vertebrate cys-loop GABA receptors, those in nematodes are quite distinct in sequence similarity, subunit composition and biological function. With rising anthelmintic resistance in many nematode populations worldwide, GABA receptors should become an area of increased scientific investigation in the development of the next generation of anthelmintics.     

Two novel loci underlie natural differences in Caenorhabditis elegans abamectin responses

Parasitic nematodes cause a massive worldwide burden on human health along with a loss of livestock and agriculture productivity. Anthelmintics have been widely successful in treating parasitic nematodes. However, resistance is increasing, and little is known about the molecular and genetic causes of resistance for most of these drugs. The free-living roundworm Caenorhabditis elegans provides a tractable model to identify genes that underlie resistance. Unlike parasitic nematodes, C. elegans is easy to maintain in the laboratory, has a complete and well annotated genome, and has many genetic tools. Using a combination of wild isolates and a panel of recombinant inbred lines constructed from crosses of two genetically and phenotypically divergent strains, we identified three genomic regions on chromosome V that underlie natural differences in response to the macrocyclic lactone (ML) abamectin. One locus was identified previously and encodes an alpha subunit of a glutamate-gated chloride channel (glc-1). Here, we validate and narrow two novel loci using near-isogenic lines. Additionally, we generate a list of prioritized candidate genes identified in C. elegans and in the parasite Haemonchus contortus by comparison of ML resistance loci. These genes could represent previously unidentified resistance genes shared across nematode species and should be evaluated in the future. Our work highlights the advantages of using C. elegans as a model to better understand ML resistance in parasitic nematodes.     

Plant parasitic nematode proteins and the host parasite interaction.

This review focuses on the proteins and secretions of sedentary plant parasitic nematodes potentially important for plant-nematode interactions. These nematodes are well equipped for parasitism of plants. Having acquired the ability to manipulate fundamental aspects of plant biology, they are able to hijack host-cell development to make their feeding site. They feed exclusively from feeding sites as they complete their life cycle, satisfying their nutritional demands for development and reproduction. Biochemical and genomic approaches have been used successfully to identify a number of nematode parasitism genes. So far, 65 204 expressed sequence tags (ESTs) have been generated for six Meloidogyne species and sequencing projects, currently in progress, will underpin genomic comparisons of Meloidogyne spp. with sequences of other pathogens and generate genechip microarrays to undertake profiling studies of up- and down-regulated genes during the infection process. RNA interference provides a molecular genetic tool to study gene function in parasitism. These methods should provide new data to help our understanding of how parasitic nematodes infect their hosts, leading to the identification of novel pathogenicity genes.     

Biological control potential of plant growth‐promoting rhizobacteria suppression of Meloidogyne incognita on cotton and Heterodera glycines on soybean: A review

Over the past decade, we have seen an increasing market for biopesticides and an increase in number of microbial control studies directed towards plant‐parasitic nematodes. This literature survey provides an overview of research on biological control of two economically important plant‐parasitic nematodes, Meloidogyne incognita (Kofoid & White) Chitwood (southern root‐knot nematode) and Heterodera glycines Ichinohe (soybean cyst nematode) using spore‐forming plant growth‐promoting rhizobacteria (PGPR). In this review, the current biological control strategies for the management of those cotton and soybean nematodes, the mechanism of using BacillusPGPR for biological control of plant‐parasitic nematode including induced systemic resistance and antagonism and the future of biological control agents on management of plant‐parasitic nematodes are covered.     

线虫耐寒性研究进展

对于分布在温带和寒带的线虫,它们只有战胜冬季寒冷的挑战,才能有利于种群的存在与发展。因此,耐寒性是线虫生物学研究中不可忽视的内容。综述了关于线虫在低温胁迫下的耐寒性测定方法、耐寒对策及耐寒机制等方面的研究进展。线虫的耐寒性和昆虫一样,可通过过冷却点和低温存活率两种指标进行评价,但在具体的实验方法上,线虫耐寒性研究有其不同之处。线虫的耐寒对策和耐寒机制具有多样化。耐寒对策主要有耐冻和避冻,二者能共同渗透于线虫的耐寒过程中。耐寒机制包括特殊发育阶段的形成、低温驯化作用、低分子量抗冻物质的聚集、以及高分子量抗冻蛋白和热休克蛋白的产生,等等。此外,还强调应从多个角度研究线虫的耐寒性,如寒冷敏感型线虫的研究、寄生线虫的耐寒对策研究以及交叉胁迫的研究。     

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 molecular diagnostic tool to replace larval culture in conventional faecal egg count reduction testing in sheep

The accurate diagnosis of parasitic nematode infections in livestock (including sheep and goats) is central to their effective control and the detection of the anthelmintic resistance. Traditionally, the faecal egg count reduction test (FECRT), combined with the technique of larval culture (LC), has been used widely to assess drug-susceptibility/resistance in strongylid nematodes. However, this approach suffers from a lack of specificity, sensitivity and reliability, and is time-consuming and costly to conduct. Here, we critically assessed a specific PCR assay to support FECRT, in a well-controlled experiment on sheep with naturally acquired strongylid infections known to be resistant to benzimidazoles. We showed that the PCR results were in close agreement with those of total worm count (TWC), but not of LC. Importantly, albendazole resistance detected by PCR-coupled FECRT was unequivocally linked to Teladorsagia circumcincta and, to lesser extent, Trichostrongylus colubriformis, a result that was not achievable by LC. The key findings from this study demonstrate that our PCR-coupled FECRT approach has major merit for supporting anthelmintic resistance in nematode populations. The findings also show clearly that our PCR assay can be used as an alternative to LC, and is more time-efficient and less laborious, which has important practical implications for the effective management and control strongylid nematodes of sheep.     

Advances in the Diagnosis of Endemic Treponematoses: Yaws,Bejel, and Pinta

Improved understanding of the differential diagnosis of endemic treponematoses is needed to inform clinical practice and to ensure the best outcome for a new global initiative for the eradication of yaws, bejel, and pinta. Traditionally, the human treponematoses have been differentiated based upon their clinical manifestations and epidemiologic characteristics because the etiologic agents are indistinguishable in the laboratory. Serological tests are still considered standard laboratory methods for the diagnosis of endemic treponematoses and new rapid point-of-care treponemal tests have become available which are extremely useful in low-resource settings. In the past ten years, there has been an increasing effort to apply polymerase chain reaction to treponematoses and whole genome fingerprinting techniques have identified genetic signatures that can differentiate the existing treponemal strains; however, definitive diagnosis is also hampered by widespread unavailability of molecular diagnostics. We review the dilemmas in the diagnosis of endemic treponematoses, and advances in the discovery of new diagnostic tools.     

Deep amplicon sequencing as a powerful new tool to screen for sequence polymorphisms associated with anthelmintic resistance in parasitic nematode populations

Parasitic gastrointestinal nematodes contribute to significant human morbidity and cause billions of dollars per year in lost agricultural production. Control is dependent on the use of anthelmintic drugs which, in the case of livestock parasites, is severely compromised by the widespread development of drug resistance. There are now concerns regarding the emergence of anthelmintic resistance in parasitic nematodes of humans in response to the selection pressure resulting from mass drug administration programs. Consequently, there is an urgent need for sensitive, scalable and accurate diagnostic tools to detect the emergence of anthelmintic resistance. Detecting and measuring the frequency of resistance-associated mutations in parasite populations has the potential to provide sensitive and quantitative assessment of resistance emergence from an early stage. Here, we describe the development and validation of deep amplicon sequencing as a powerful new approach to detect and quantify the frequency of single nucleotide polymorphisms associated with benzimidazole resistance. We have used parasite communities in sheep to undertake a proof-of-concept study of this approach. Sheep provide an excellent host system, as there are multiple co-infecting trichostrongylid nematode species, each likely with a varying prevalence of benzimidazole resistance. We demonstrate that the approach provides an accurate measure of resistance allele frequencies, and can reliably detect resistance alleles down to a frequency of 0.1%, making it particularly valuable for screening mutations in the early stages of resistance. We illustrate the power of the technique by screening UK sheep flocks for benzimidazole resistance-associated single nucleotide polymorphisms at three different codons of the β-tubulin gene in seven different parasite species from 164 populations (95 from ewes and 69 from lambs) in a single MiSeq sequencing run. This approach provides a powerful new tool to screen for the emergence of anthelmintic resistance mutations in parasitic nematode populations of both animals and humans.