From parasite genomes to one healthy world: Are we having fun yet?

In 1990, the Human Genome Sequencing Project was established. This laid the ground work for an explosion of sequence data that has since followed. As a result of this effort, the first complete genome of an animal, Caenorhabditis elegans was published in 1998. The sequence of Drosophila melanogaster was made available in March, 2000 and in the following year, working drafts of the human genome were generated with the completed sequence (92%) being released in 2003. Recent advancements and next-generation technologies have made sequencing common place and have infiltrated every aspect of biological research, including parasitology. To date, sequencing of 32 apicomplexa and 24 nematode genomes are either in progress or near completion, and over 600k nematode EST and 200k apicomplexa EST submissions fill the databases. However, the winds have shifted and efforts are now refocusing on how best to store, mine and apply these data to problem solving. Herein we tend not to summarize existing X-omics datasets or present new technological advances that promise future benefits. Rather, the information to follow condenses up-to-date-applications of existing technologies to problem solving as it relates to parasite research. Advancements in non-parasite systems are also presented with the proviso that applications to parasite research are in the making

Immunological responses in the mouse host to a cloned antigen of Taeniacrassiceps

Adult female Swiss-Webster mice were immunized either intraperitoneally (IP) or subcutaneously (SQ) with cyst fluid or a genetically engineered fusion protein, Taenia carassiceps antigen 2-maltose binding protein (TCA2-MBP) from Taenia crassiceps metacestodes, or with live, non-budding cysts SQ, and then challenged IP with T. crassiceps metacestodes and necropsied 9 weeks later. Numbers of peripheral blood eosinophils were increased after IP immunization, but were not increased after SQ immunization or with SQ cysts given before the challenge infection. Eosinophil numbers gradually decreased over the course of the experiment, and were not found in increased numbers in the blood or peritoneal cavity at necropsy. Antigen-specific antibody responses were seen at day 14 or 28 in IP and SQ immunized groups; IgG3 and IgG3 isotypes continued to increase over the course of the experiment. A significant protective response was induced by immunization with the cyst fluid (15 + 4, X + SE recovered larvae) or the TCA2-MBP (22+12) given IP, but not SQ (122+36l ; 207+53, respectively) as measured by the numbers of larvae recovered at necropsy. Live cysts given SQ resulted in reduced numbers of cysts in the peritoneal cavity (188+66), but was not as effective as cyst fluid or TCA2-MBP given IP. Locally (IP) induced immune responses may be involved in the development of the protective response to a challenge infection with T. crassiceps metacestodes

Integrating genomics and phylogenetics in understanding the history of Trichinella species

In 2004, funding was received by Washington University’s Genome Sequencing Center through NHGRI, to completely sequence several nematode genomes as part of a holistic effort to advance our understanding of the human genome and evolution within the Metazoa. Trichinella spiralis was among this group of worms because of its strategic location at the base of the phylum Nematoda, and the belief that extant species represented an ancient divergent event that occurred as early as the Paleozoic. At the same time, a concerted effort was put forth to solidify the phylogeny of extant species of Trichinella based upon molecular analyses of a multi-gene system to understand the history of the genus and thereby enhance utilization of the forthcoming sequence data. Since the inception of this research, several findings have emerged: (1) the size of T. spiralis genome estimated by flow cytometry (71.3 Mb) is substantially smaller than originally predicted (270 Mb); (2) to date, a subset of the total of 3,534,683 sequences have been assembled into a 59.3 Mb unique sequence; (3) 19% of the assembled sequence is comprised of repetitive elements; and (4) sequence data are predicated upon extant T. spiralis which probably diverged as little as 20 million years ago. Thus, the utility of the T. spiralis genome as representative of an archaic species must be tempered with the knowledge that encapsulated and non-encapsulated clades probably separated during the mid-Miocene as temperate ecosystems changed

International Commission on Trichinellosis: Recommendations for genotyping Trichinella muscle stage larvae

Being able to identify the species or genotype of Trichinella is of paramount importance not only for epidemiological studies but to better ascertain the source of outbreaks that still occur worldwide. This has become more critical in recent years given the increase in imported meat products and the relationship that wild animals play in the domestic and sylvatic transmission cycles. In contrast to a time when the genus Trichinella was considered monospecific, research in recent years has revealed that the genus consists of 9 species and at least 3 additional genotypes which have yet to be named. Except for a non-encapsulated clade consisting of Trichinella pseudospiralis, Trichinella zimbabwensis, and Trichinella papuae, all members of this genus are morphologically indistinguishable. Thus, identification has been relegated to using PCR and in special cases, DNA sequencing or restriction enzyme digestion. Rather than using a collection of PCR primers specific for each genotype, a single multiplex PCR previously developed for differentiating the major encapsulated and non-encapsulated genotypes has been adopted by the International Commission on Trichinellosis. Since the assay was first developed, other species have been named. Thus, DNA sequencing has been used to validate closely related genotypes. The ICT recommends genotyping be performed as described herein during all outbreaks and whenever Trichinella has been found in consumable foods

A multiplex polymerase chain reaction assay to simultaneously distinguish Cryptosporidium species of veterinary and public health concern in cattle

Four species of Cryptosporidium are routinely found in cattle: Cryptosporidium parvum, Cryptosporidium bovis, Cryptosporidium ryanae, and Cryptosporidium andersoni. It is important to determine the species of Cryptosporidium in infected cattle because C. parvum is the only serious pathogen for humans as well as cattle. Identification of Cryptosporidium species and genotypes currently relies on molecular methods such as polymerase chain reaction (PCR) followed by restriction fragment length polymorphism (RFLP) or gene sequencing. Incorporation of these techniques in a routine veterinary diagnostic laboratory is cost prohibitive. As such, their applications are limited primarily to research and a few public health laboratories. To overcome this problem, a multiplex PCR assay was developed for simultaneously detecting the 4 species of Cryptosporidium that commonly infect cattle. This assay specifically identifies Cryptosporidium oocysts present in cattle feces, improves the detection of mixed infections, reduces the time and cost relative to current sequencing methods, and further demonstrates the shortcomings of sequencing as the definitive method for identification when analyzing samples containing mixed infections

PCR as a diagnostic and quantitative technique in veterinary parasitology

Over the past 15 years, there has been a dramatic evolution in molecular approaches to study parasites and parasitic diseases. Many of these advancements have been brought about through the development of new applications of the polymerase chain reaction (PCR). Enhancements in sensitivity that can be achieved using PCR now permit scientists to investigate changes at the level of a single cell, far below what is often needed for parasite-derived applications. PCR has had a substantial impact on advances made in the areas of parasite systematics and epidemiology, immunology and host–parasite interactions, recombinant DNA vaccine development and most re- cently, the analysis of whole genomes either through directly sequencing the DNA, the analysis of expressed sequence tags (ESTs) or through the rapidly growing field of functional genomics. This paper, however, focuses on the application of PCR methodology to parasite detection and differen- tiation, and the diagnosis of disease. Specific attention is given to advances provided by multiplex PCR, fluorescence-based “real-time” PCR, and the utilization of PCR as a quantitative technique. Published by Elsevier Science B.V

Transmissible gastroenteritis virus: Identification of M protein-binding peptide ligands with antiviral and diagnostic potential

The membrane (M) protein is one of the major structural proteins of coronavirus particles. In this study, the M protein of transmissible gastroenteritis virus (TGEV) was used to biopan a 12-mer phage display random peptide library. Three phages expressing TGEV-M-binding peptides were identified and characterized in more depth. A phage-based immunosorbent assay (phage-ELISA) capable of differentiating TGEV from other coronaviruses was developed using one phage, phTGEV-M7, as antigen. When the phage-ELISA was compared to conventional antibody-based ELISA for detecting infections, phage-ELISA exhibited greater sensitivity. A chemically synthesized, TGEV-M7 peptide (pepTGEV-M7; HALTPIKYIPPG) was evaluated for antiviral activity. Plaque-reduction assays revealed that pepTGEV-M7 was able to prevent TGEV infection in vitro (p \u3c 0.01) following pretreatment of the virus with the peptide. Indirect immunofluorescence and real-time RT-PCR confirmed the inhibitory effects of the peptide. These results indicate that pepTGEV-M7 might be utilized for virus-specific diagnostics and treatment

Trichinella spiralis: Adaptation and parasitism

Publication of the genome from the clade I organism, Trichinella spiralis, has provided us an avenue to address more holistic problems in parasitology; namely the processes of adaptation and the evolution of parasitism. Parasitism among nematodes has evolved in multiple, independent events. Deciphering processes that drive species diversity and adaptation are keys to understanding parasitism and advancing control strategies. Studies have been put forth on morphological and physiological aspects of parasitism and adaptation in nematodes; however, data is now coming available to investigate adaptation, host switching and parasitism at the genomic level. Herein we compare proteomic data from the clade I parasite, Trichinella spiralis with data from Brugia malayi (clade III), Meloidogyne hapla and Meloidogyne incognita (clade IV), and free-living nematodes belonging to the genera Caenorhabditis and Pristionchus (clade V). We explore changes in protein family birth/death and expansion/reduction over the course of metazoan evolution using Homo sapiens, Drosophila melanogaster and Saccharomyces cerevisiae as out- groups for the phylum Nematoda. We further examine relationships between these changes and the ability and/or result of nematodes adapting to their environments. Data are consistent with gene loss occurring in conjunction with nematode specialization resulting from parasitic worms acclimating to well-defined, environmental niches. We observed evidence for independent, lateral gene transfer events involving conserved genes that may have played a role in the evolution of nematode parasitism. In general, parasitic nematodes gained proteins through duplication and lateral gene transfer, and lost proteins through random mutation and deletions. Data suggest independent acquisition rather than ancestral inheritance among the Nematoda followed by selective gene loss over evolutionary time. Data also show that parasitism and adaptation affected a broad range of proteins, especially those involved in sensory perception, metabolism, and transcription/translation. New protein gains with functions related to regu- lating transcription and translation, and protein family expansions with functions related to morphology and body development have occurred in association with parasitism. Further gains occurred as a result of lateral gene transfer and in particular, with the cyanase protein family In contrast, reductions and/or losses have occurred in protein families with functions related to metabolic process and signal trans- duction. Taking advantage of the independent occurrences of parasitism in nematodes, which enabled us to distinguish changes associated with parasitism from species specific niche adaptation, our study provides valuable insights into nematode parasitism at a proteome level using T. spiralis as a benchmark for early adaptation to or acquisition of parasitism

The Identification of Haemonchus Species and Diagnosis of Haemonchosis

Diagnosis is often equated with identification or detection when discussing parasitic diseases. Unfortunately, these are not necessarily mutually exclusive activities; diseases and infections are generally diagnosed and organisms are identified. Diagnosis is commonly predicated upon some clinical signs; in an effort to determine the causative agent, identification of genera and species is subsequently performed. Both identification and diagnosis play critical roles in managing an infection, and involve the interplay of direct and indirect methods of detection, particularly in light of the complex and expanding problem of drug-resistance in parasites. Accurate and authoritative identification that is cost- and time-effective, based on structural and molecular attributes of specimens, provides a foundation for defining parasite diversity and changing patterns of geographical distribution, host association and emergence of disease. Most techniques developed thus far have been grounded in assumptions based on strict host associations between Haemonchus contortus and small ruminants, that is, sheep and goats, and between Haemonchus placei and bovids. Current research and increasing empirical evidence of natural infections in the field demonstrates that this assumption misrepresents the host associations for these species of Haemonchus. Furthermore, the capacity of H. contortus to utilize a considerably broad spectrum of ungulate hosts is reflected in our understanding of the role of anthropogenic forcing, the ‘breakdown’ of ecological isolation, global introduction and host switching as determinants of dis- tribution. Nuanced insights about distribution, host association and epidemiology have emerged over the past 30 years, coincidently with the development of increasingly robust means for parasite identification. In this review and for the sake of argument, we would like to delineate the diagnosis of haemonchosis from the identification of the specific pathogen. As a foundation for exploring host and parasite biology, we will examine the evolution of methods for distinguishing H. contortus from other com- mon gastrointestinal nematodes of agriculturally significant and free-ranging wild ru- minants using morphological, molecular and/or immunological methods for studies at the species and genus level

Immunological responses in the mouse host to a cloned antigen of Taeniacrassiceps

Adult female Swiss-Webster mice were immunized either intraperitoneally (IP) or subcutaneously (SQ) with cyst fluid or a genetically engineered fusion protein, Taenia carassiceps antigen 2-maltose binding protein (TCA2-MBP) from Taenia crassiceps metacestodes, or with live, non-budding cysts SQ, and then challenged IP with T. crassiceps metacestodes and necropsied 9 weeks later. Numbers of peripheral blood eosinophils were increased after IP immunization, but were not increased after SQ immunization or with SQ cysts given before the challenge infection. Eosinophil numbers gradually decreased over the course of the experiment, and were not found in increased numbers in the blood or peritoneal cavity at necropsy. Antigen-specific antibody responses were seen at day 14 or 28 in IP and SQ immunized groups; IgG3 and IgG3 isotypes continued to increase over the course of the experiment. A significant protective response was induced by immunization with the cyst fluid (15 + 4, X + SE recovered larvae) or the TCA2-MBP (22+12) given IP, but not SQ (122+36l ; 207+53, respectively) as measured by the numbers of larvae recovered at necropsy. Live cysts given SQ resulted in reduced numbers of cysts in the peritoneal cavity (188+66), but was not as effective as cyst fluid or TCA2-MBP given IP. Locally (IP) induced immune responses may be involved in the development of the protective response to a challenge infection with T. crassiceps metacestodes