Characterization of viable but nonculturable state of Campylobacter concisus.

Campylobacter concisus is an opportunistic bacterial pathogen linked with a range of human diseases. The objective of this study was to investigate the viable but nonculturable (VBNC) state of the bacterium. To induce the VBNC state, C. concisus cells were maintained in sterilized phosphate-buffered saline at 4°C for three weeks. The VBNC cells were monitored using quantitative analysis by propidium monoazide (PMAxx) coupled with quantitative real-time PCR (PMAxx-qPCR), targeting the DNA gyrase subunit B gene. The results demonstrated that C. concisus ATCC 51562 entered the VBNC state in 15 days, while ATCC 51561 entered the VBNC state in 9 days. The viable cell counts, assessed by PMAxx-qPCR, consistently remained close to the initial level of 107 CFU ml-1, indicating a substantial portion of the cell population had entered the VBNC state. Notably, morphological analysis revealed that the VBNC cells became coccoid and significantly smaller. The cells could be resuscitated through a temperature increase in the presence of a highly nutritious growth medium. In conclusion, under environmental stress, most C. concisus cells converted to the VBNC state. The VBNC state of C. concisus may be important for its environmental survival and spread, and the presence of VBNC forms should be considered in environmental and clinical monitoring.

Draft genome of the bluefin tuna blood fluke, Cardicola forsteri.

The blood fluke Cardicola forsteri (Trematoda: Aporocotylidae) is a pathogen of ranched bluefin tuna in Japan and Australia. Genomics of Cardicola spp. have thus far been limited to molecular phylogenetics of select gene sequences. In this study, sequencing of the C. forsteri genome was performed using Illumina short-read and Oxford Nanopore long-read technologies. The sequences were assembled de novo using a hybrid of short and long reads, which produced a high-quality contig-level assembly (N50 > 430 kb and L50 = 138). The assembly was also relatively complete and unfragmented, comprising 66% and 7.2% complete and fragmented metazoan Benchmarking Universal Single-Copy Orthologs (BUSCOs), respectively. A large portion (> 55%) of the genome was made up of intergenic repetitive elements, primarily long interspersed nuclear elements (LINEs), while protein-coding regions cover > 6%. Gene prediction identified 8,564 hypothetical polypeptides, > 77% of which are homologous to published sequences of other species. The identification of select putative proteins, including cathepsins, calpains, tetraspanins, and glycosyltransferases is discussed. This is the first genome assembly of any aporocotylid, a major step toward understanding of the biology of this family of fish blood flukes and their interactions within hosts.

Enhancement of live vaccines by co-delivery of immune modulating proteins.

Vaccines are very effective in providing protection against many infectious diseases. However, it has proven difficult to develop highly efficacious vaccines against some pathogens and so there is a continuing need to improve vaccine technologies. The first successful and widely used vaccines were based on attenuated pathogens (e.g., laboratory passaged Pasteurella multocida to vaccinate against fowl cholera) or closely related non-pathogenic organisms (e.g., cowpox to vaccinate against smallpox). Subsequently, live vaccines, either attenuated pathogens or non-pathogenic microorganisms modified to deliver heterologous antigens, have been successfully used to induce protective immune responses against many pathogens. Unlike conventional killed and subunit vaccines, live vaccines can deliver antigens to mucosal surfaces in a similar manner and context as the natural infection and hence can often produce a more appropriate and protective immune response. Despite these advantages, there is still a need to improve the immunogenicity of some live vaccines. The efficacy of injectable killed and subunit vaccines is usually enhanced using adjuvants such mineral salts, oils, and saponin, but such adjuvants cannot be used with live vaccines. Instead, live vaccines can be engineered to produce immunomodulatory molecules that can stimulate the immune system to induce more robust and long-lasting adaptive immune responses. This review focuses on research that has been undertaken to engineer live vaccines to produce immunomodulatory molecules that act as adjuvants to increase immunogenicity. Adjuvant strategies with varying mechanisms of action (inflammatory, antibody-mediated, cell-mediated) and delivery modes (oral, intramuscular, intranasal) have been investigated, with varying degrees of success. The goal of such research is to define adjuvant strategies that can be adapted to enhance live vaccine efficacy by triggering strong innate and adaptive immune responses and produce vaccines against a wider range of pathogens.

Impact of next-generation technologies on exploring socioeconomically important parasites and developing new interventions.

High-throughput molecular and computer technologies have become instrumental for systems biological explorations of pathogens, including parasites. For instance, investigations of the transcriptomes of different developmental stages of parasitic nematodes give insights into gene expression, regulation and function in a parasite, which is a significant step to understanding their biology, as well as interactions with their host(s) and disease. This chapter (1) gives a background on some key parasitic nematodes of socioeconomic importance, (2) describes sequencing and bioinformatic technologies for large-scale studies of the transcriptomes and genomes of these parasites, (3) provides some recent examples of applications and (4) emphasizes the prospects of fundamental biological explorations of parasites using these technologies for the development of new interventions to combat parasitic diseases.

The genome and developmental transcriptome of the strongylid nematode Haemonchus contortus.

BACKGROUND: The barber's pole worm, Haemonchus contortus, is one of the most economically important parasites of small ruminants worldwide. Although this parasite can be controlled using anthelmintic drugs, resistance against most drugs in common use has become a widespread problem. We provide a draft of the genome and the transcriptomes of all key developmental stages of H. contortus to support biological and biotechnological research areas of this and related parasites. RESULTS: The draft genome of H. contortus is 320 Mb in size and encodes 23,610 protein-coding genes. On a fundamental level, we elucidate transcriptional alterations taking place throughout the life cycle, characterize the parasite's gene silencing machinery, and explore molecules involved in development, reproduction, host-parasite interactions, immunity, and disease. The secretome of H. contortus is particularly rich in peptidases linked to blood-feeding activity and interactions with host tissues, and a diverse array of molecules is involved in complex immune responses. On an applied level, we predict drug targets and identify vaccine molecules. CONCLUSIONS: The draft genome and developmental transcriptome of H. contortus provide a major resource to the scientific community for a wide range of genomic, genetic, proteomic, metabolomic, evolutionary, biological, ecological, and epidemiological investigations, and a solid foundation for biotechnological outcomes, including new anthelmintics, vaccines and diagnostic tests. This first draft genome of any strongylid nematode paves the way for a rapid acceleration in our understanding of a wide range of socioeconomically important parasites of one of the largest nematode orders.

Whole-genome sequence of Schistosoma haematobium.

Schistosomiasis is a neglected tropical disease caused by blood flukes (genus Schistosoma; schistosomes) and affecting 200 million people worldwide. No vaccines are available, and treatment relies on one drug, praziquantel. Schistosoma haematobium has come into the spotlight as a major cause of urogenital disease, as an agent linked to bladder cancer and as a predisposing factor for HIV/AIDS. The parasite is transmitted to humans from freshwater snails. Worms dwell in blood vessels and release eggs that become embedded in the bladder wall to elicit chronic immune-mediated disease and induce squamous cell carcinoma. Here we sequenced the 385-Mb genome of S. haematobium using Illumina-based technology at 74-fold coverage and compared it to sequences from related parasites. We included genome annotation based on function, gene ontology, networking and pathway mapping. This genome now provides an unprecedented resource for many fundamental research areas and shows great promise for the design of new disease interventions.

Key strongylid nematodes of animals - Impact of next-generation transcriptomics on systems biology and biotechnology.

The advent and integration of high-throughput 'omic technologies (e.g., genomics, transcriptomics, proteomics and metabolomics) are becoming instrumental to assist fundamental explorations of the systems biology of organisms. In particular, these technologies now provide unique opportunities for global, molecular investigations of parasites. For example, studies of the transcriptomes (all transcripts in an organism, tissue or cell) of different species and/or developmental stages of parasitic nematodes provide insights into aspects of gene expression, regulation and function, which is a major step to understanding their biology. The purpose of this article was to review salient aspects of the systematics and biology of selected species of parasitic nematodes (particularly key species of the order Strongylida) of socio-economic importance, to describe conventional and advanced sequencing technologies and bioinformatic tools for large-scale investigations of the transcriptomes of these parasites and to highlight the prospects and implications of these explorations for developing novel methods of parasite intervention.

Detection of diarrhoeal pathogens in human faeces using an automated, robotic platform.

Infectious diarrhoeal diseases represent a major socio-economic burden to humans, and are linked to a range of pathogens, including viruses, bacteria and protists. The accurate detection of such pathogens is central to control. However, detection often relies on methods that have limited diagnostic sensitivity and specificity. Here, we assessed an automated, robotic platform for the simultaneous detection of eight major pathogens associated with infectious diarrhoea. Genomic DNA samples (n = 167) from faeces from humans with diarrhoea and diagnosed as cryptosporidiosis, and 100 uninfected control subjects, were tested for adenovirus 40/41, norovirus, Clostridium difficile, Campylobacter, Salmonella, Shigella, Cryptosporidium and Giardia by multiplexed-tandem PCR, and also characterized by single-strand conformation polymorphism analysis (SSCP) and selective sequencing. All 167 samples tested positive for Cryptosporidium, five for adenovirus 40/41, four for Campylobacter, three for C. difficile and seven for Shigella spp., with no false positive results for any assay. The automated PCR exhibited a high sensitivity, with <10 individual pathogens being readily detected. The robotic detection platform assessed here represents a sensitive, high-throughput tool for key pathogens linked to infectious diarrhoea in humans. This platform requires little molecular biological expertise and is well suited to various diagnostic facilities and settings.

Cryptic parasite revealed improved prospects for treatment and control of human cryptosporidiosis through advanced technologies.

Cryptosporidium is an important genus of parasitic protozoa of humans and other vertebrates and is a major cause of intestinal disease globally. Unlike many common causes of infectious enteritis, there are no widely available, effective vaccine or drug-based intervention strategies for Cryptosporidium, and control is focused mainly on prevention. This approach is particularly deficient for infections of severely immunocompromised and/or suppressed, the elderly or malnourished people. However, cryptosporidiosis also presents a significant burden on immunocompetent individuals, and can, for example have lasting effects on the physical and mental development of children infected at an early age. In the last few decades, our understanding of Cryptosporidium has expanded significantly in numerous areas, including the parasite life-cycle, the processes of excystation, cellular invasion and reproduction, and the interplay between parasite and host. Nonetheless, despite extensive research, many aspects of the biology of Cryptosporidium remain unknown, and treatment and control are challenging. Here, we review the current state of knowledge of Cryptosporidium, with a focus on major advances arising from the recently completed genome sequences of the two species of greatest relevance in humans, namely Cryptosporidium hominis and Cryptosporidium parvum. In addition, we discuss the potential of next-generation sequencing technologies, new advances in in silico analyses and progress in in vitro culturing systems to bridge these gaps and to lead toward effective treatment and control of cryptosporidiosis.

Ascaris suum draft genome.

Parasitic diseases have a devastating, long-term impact on human health, welfare and food production worldwide. More than two billion people are infected with geohelminths, including the roundworms Ascaris (common roundworm), Necator and Ancylostoma (hookworms), and Trichuris (whipworm), mainly in developing or impoverished nations of Asia, Africa and Latin America. In humans, the diseases caused by these parasites result in about 135,000 deaths annually, with a global burden comparable with that of malaria or tuberculosis in disability-adjusted life years. Ascaris alone infects around 1.2 billion people and, in children, causes nutritional deficiency, impaired physical and cognitive development and, in severe cases, death. Ascaris also causes major production losses in pigs owing to reduced growth, failure to thrive and mortality. The Ascaris-swine model makes it possible to study the parasite, its relationship with the host, and ascariasis at the molecular level. To enable such molecular studies, we report the 273 megabase draft genome of Ascaris suum and compare it with other nematode genomes. This genome has low repeat content (4.4%) and encodes about 18,500 protein-coding genes. Notably, the A. suum secretome (about 750 molecules) is rich in peptidases linked to the penetration and degradation of host tissues, and an assemblage of molecules likely to modulate or evade host immune responses. This genome provides a comprehensive resource to the scientific community and underpins the development of new and urgently needed interventions (drugs, vaccines and diagnostic tests) against ascariasis and other nematodiases.

The transcriptome of Trichuris suis--first molecular insights into a parasite with curative properties for key immune diseases of humans.

BACKGROUND: Iatrogenic infection of humans with Trichuris suis (a parasitic nematode of swine) is being evaluated or promoted as a biological, curative treatment of immune diseases, such as inflammatory bowel disease (IBD) and ulcerative colitis, in humans. Although it is understood that short-term T. suis infection in people with such diseases usually induces a modified Th2-immune response, nothing is known about the molecules in the parasite that induce this response. METHODOLOGY/PRINCIPAL FINDINGS: As a first step toward filling the gaps in our knowledge of the molecular biology of T. suis, we characterised the transcriptome of the adult stage of this nematode employing next-generation sequencing and bioinformatic techniques. A total of ∼65,000,000 reads were generated and assembled into ∼20,000 contiguous sequences ( = contigs); ∼17,000 peptides were predicted and classified based on homology searches, protein motifs and gene ontology and biological pathway mapping. CONCLUSIONS: These analyses provided interesting insights into a number of molecular groups, particularly predicted excreted/secreted molecules (n = 1,288), likely to be involved in the parasite-host interactions, and also various molecules (n = 120) linked to chemokine, T-cell receptor and TGF-ß signalling as well as leukocyte transendothelial migration and natural killer cell-mediated cytotoxicity, which are likely to be immuno-regulatory or -modulatory in the infected host. This information provides a conceptual framework within which to test the immunobiological basis for the curative effect of T. suis infection in humans against some immune diseases. Importantly, the T. suis transcriptome characterised herein provides a curated resource for detailed studies of the immuno-molecular biology of this parasite, and will underpin future genomic and proteomic explorations.

Bovine theileriosis--an emerging problem in south-eastern Australia?

Bovine theileriosis is an arthropod-borne disease caused by one or more haemoprotozoan parasites of the genus Theileria. Traditionally, Theileria infection in cattle in Australia was largely asymptomatic and recognized to be associated with Theileria buffeli, now assigned to the Theileria orientalis-group. There have been some recent outbreaks of theileriosis in dairy and beef cattle, mainly in subtropical climatic zone (New South Wales) of Australia. Here, we provide the first published evidence of an outbreak of bovine theileriosis in the south-eastern Australia (state of Victoria) linked to the ikeda and chitose genotypes of T. orientalis. Future investigations should focus sharply on the elucidating the epidemiology and ecology of Theileria in this region to subvert the possible impact on the cattle industry.

Norcantharidin analogues with nematocidal activity in Haemonchus contortus.

With the major problems with resistance in parasitic nematodes of livestock to anthelmintic drugs, there is an urgent need to develop new nematocides. In the present study, we employed a targeted approach for the design of a series of norcantharidin analogues (n=54) for activity testing against the barber's pole worm (Haemonchus contortus) of small ruminants in a larval development assay (LDA) and also for toxicity testing on nine distinct human cell lines. Although none of the 54 analogues synthesized were toxic to any of these cell lines, three of them (N-octyl-7-oxabicyclo(2.2.1)heptane-2,3-dicarboximide (B2), N-decyl-7-oxabicyclo(2.2.1)heptane-2,3-dicarboximide (B3) and 4-[(4-methyl)-3-ethyl-2-methyl-5-phenylfuran-10-oxa-4-azatricyclo[5.2.1]decane-3,5-dione (B21) reproducibly displayed 99-100% lethality to H. contortus in LDA, with LD(50s) of 25-40 µM. The high 'hit rate' (5.6%) indicates that the approach taken here has advantages over conventional drug screening methods. A major advantage of norcantharidin analogues over some other currently available anthelmintics is that they can be produced in one to two steps in large amounts at low cost and high purity, and do not require any additional steps for the isolation of the active isomer. This positions them well for commercial development.

Major prospects for exploring canine vector borne diseases and novel intervention methods using 'omic technologies.

Canine vector-borne diseases (CVBDs) are of major socioeconomic importance worldwide. Although many studies have provided insights into CVBDs, there has been limited exploration of fundamental molecular aspects of most pathogens, their vectors, pathogen-host relationships and disease and drug resistance using advanced, 'omic technologies. The aim of the present article is to take a prospective view of the impact that next-generation, 'omics technologies could have, with an emphasis on describing the principles of transcriptomic/genomic sequencing as well as bioinformatic technologies and their implications in both fundamental and applied areas of CVBD research. Tackling key biological questions employing these technologies will provide a 'systems biology' context and could lead to radically new intervention and management strategies against CVBDs.

A portrait of the transcriptome of the neglected trematode, Fasciola gigantica--biological and biotechnological implications.

Fasciola gigantica (Digenea) is an important foodborne trematode that causes liver fluke disease (fascioliasis) in mammals, including ungulates and humans, mainly in tropical climatic zones of the world. Despite its socioeconomic impact, almost nothing is known about the molecular biology of this parasite, its interplay with its hosts, and the pathogenesis of fascioliasis. Modern genomic technologies now provide unique opportunities to rapidly tackle these exciting areas. The present study reports the first transcriptome representing the adult stage of F. gigantica (of bovid origin), defined using a massively parallel sequencing-coupled bioinformatic approach. From >20 million raw sequence reads, >30,000 contiguous sequences were assembled, of which most were novel. Relative levels of transcription were determined for individual molecules, which were also characterized (at the inferred amino acid level) based on homology, gene ontology, and/or pathway mapping. Comparisons of the transcriptome of F. gigantica with those of other trematodes, including F. hepatica, revealed similarities in transcription for molecules inferred to have key roles in parasite-host interactions. Overall, the present dataset should provide a solid foundation for future fundamental genomic, proteomic, and metabolomic explorations of F. gigantica, as well as a basis for applied outcomes such as the development of novel methods of intervention against this neglected parasite.

Evaluation and application of a molecular method to assess the composition of strongylid nematode populations in sheep with naturally acquired infections.

We evaluated the performance of a PCR method for the diagnosis of naturally acquired strongylid nematode infections in sheep (n = 470; in a temperate climatic zone of south-eastern Australia), using a panel of 100 'negative control' samples from sheep known not to harbour parasitic helminths. We compared the diagnostic sensitivity (98%) and specificity (100%) of this assay against a conventional faecal flotation method and also established a system to rank the contribution of particular strongylid nematodes to the faecal egg counts (FECs) from 'mixed infections' in individual sheep. The testing of faecal samples herein revealed that Teladorsagia circumcincta (80%) and Trichostrongylus spp. (66%) were most prevalent, followed by Chabertia ovina (33%), Oesophagostomum venulosum (28%) and Haemonchus contortus (1%). For the majority of sheep in this study, T. circumcincta and Trichostrongylus spp. represented the largest proportion of strongylid eggs in faecal samples from individual sheep. This is the first large-scale prevalence survey of gastrointestinal nematodes in live sheep using a molecular tool. The ability to rapidly rank strongylid nematodes according to their contribution to mixed infections represents a major advantage over routine coprological methods. This PCR tool has the potential to replace the conventional technique of larval culture. Future efforts will focus on enhancing and adapting this molecular method for high throughput application in routine, diagnostic settings.

Atypical (RIO) protein kinases from Haemonchus contortus--promise as new targets for nematocidal drugs.

Almost nothing is known about atypical kinases in multicellular organisms, including parasites. Supported by information and data available for the free-living nematode, Caenorhabditis elegans, and other eukaryotes, the present article describes three RIO kinase genes, riok-1, riok-2 and riok-3, from Haemonchus contortus, one of the most important parasitic nematodes of small ruminants. Analyses of these genes and their products predict that they each play critical roles in the developmental pathways of parasitic nematodes. The findings of this review indicate prospects for functional studies of these genes in C. elegans (as a surrogate) and opportunities for the design of a novel class of nematode-specific inhibitors of RIO kinases. The latter aspect is of paramount importance, given the serious problems linked to anthelmintic resistance in parasitic nematode populations of livestock.

Serine/threonine phosphatases in socioeconomically important parasitic nematodes--prospects as novel drug targets?

Little is known about the fundamental biology of parasitic nematodes (=roundworms) that cause serious diseases, affecting literally billions of animals and humans worldwide. Unlocking the biology of these neglected pathogens using modern technologies will yield crucial and profound knowledge of their molecular biology, and could lead to new treatment and control strategies. Supported by studies in the free-living nematode, Caenorhabditis elegans, some recent investigations have provided improved insights into selected protein phosphatases (PPs) of economically important parasitic nematodes (Strongylida). In the present article, we review this progress and assess the potential of serine/threonine phosphatase (STP) genes and/or their products as targets for new nematocidal drugs. Current information indicates that some small molecules, known to specifically inhibit PPs, might be developed as nematocides. For instance, some cantharidin analogues are known to display exquisite PP-inhibitor activity, which indicates that some of them could be designed and tailored to specifically inhibit selected STPs of nematodes. This information provides prospects for the discovery of an entirely novel class of nematocides, which is of paramount importance, given the serious problems linked to anthelmintic resistance in parasitic nematode populations of livestock, and has the potential to lead to significant biotechnological outcomes.

A first insight into the genotypes of Echinococcus granulosus from humans in Mongolia.

Polymerase chain reaction (PCR)-based single-strand conformation polymorphism (SSCP) and targeted sequencing were employed to genetically classify Echinococcus granulosus cysts from humans from 12 provinces in Mongolia using two DNA loci, designated pcox-1 and pnad-1, within the mitochondrial cytochrome c oxidase subunit 1 (cox-1) and NADH dehydrogenase subunit 1 (nad-1) genes, respectively. SSCP analysis of pcox-1 and pnad-1 amplicons produced from genomic DNA samples from individual E. granulosus cysts (n = 50) from individual humans displayed four distinct electrophoretic profiles for each pcox-1 and pnad-1. The direct sequencing of selected amplicons representing each of these profiles defined four distinct sequence types for each locus, present in four different combinations (designated as haplotypes M1-M4) for all 50 cyst isolates. Phylogenetic analysis of concatenated sequence data for these four haplotypes, including well-defined reference sequences, inferred that 68% of the cyst isolates belonged to the G1-G3 complex of E. granulosus (or E. granulosus sensu stricto), whereas the remaining (32%) were linked to the G6-G10 complex (or Echinococcus canadensis). Humans infected with E. granulosus cysts of the G1-G3 complex originated mainly from the eastern regions of Mongolia, whereas those harbouring cysts of the G6-G10 complex were from the western part of this country. The present study provides a first glimpse of the genetic composition of E. granulosus from humans in Mongolia, and forms a foundation for future studies of the epidemiology and ecology of the parasite(s) in animals and humans in this and surrounding countries.

Deep insights into Dictyocaulus viviparus transcriptomes provides unique prospects for new drug targets and disease intervention.

The lungworm, Dictyocaulus viviparus, causes parasitic bronchitis in cattle, and is responsible for substantial economic losses in temperate regions of the world. Here, we undertake the first large-scale exploration of available transcriptomic data for this lungworm, examine differences in transcription between different stages/both genders and identify and prioritize essential molecules linked to fundamental metabolic pathways, which could represent novel drug targets. Approximately 3 million expressed sequence tags (ESTs), generated by 454 sequencing from third-stage larvae (L3s) as well as adult females and males of D. viviparus, were assembled and annotated. The assembly of these sequences yielded ~61,000 contigs, of which relatively large proportions encoded collagens (4.3%), ubiquitins (2.1%) and serine/threonine protein kinases (1.9%). Subtractive analysis in silico identified 6928 nucleotide sequences as being uniquely transcribed in L3, and 5203 and 7889 transcripts as being exclusive to the adult female and male, respectively. Most peptides predicted from the conceptual translations were nucleoplasmins (L3), serine/threonine protein kinases (female) and major sperm proteins (male). Additional analyses allowed the prediction of three drug target candidates, whose Caenorhabditis elegans homologues were linked to a lethal RNA interference phenotype. This detailed exploration, combined with future transcriptomic sequencing of all developmental stages of D. viviparus, will facilitate future investigations of the molecular biology of this parasitic nematode as well as genomic sequencing. These advances will underpin the discovery of new drug and/or vaccine targets, focused on biotechnological outcomes.