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

Post-Revolutionary Effects: Political Self-Education of Tunisian Youth

Tunisia is a country with a rich and diverse historical and cultural background that has absorbed many ideas from western thought into its political and educational systems. For many years, the Tunisian “Republic” had the appearance of a government similar to Western democracies, yet the president’s actions were very far from those of a democratically elected president. The flaws in former president Zine El Abidine Ben Ali (Ben Ali)’s government in addition to sweeping unemployment and underdevelopment were the factors which led to the major uprising that became the Tunisian revolution. Although the revolution was a great symbolic victory for the Tunisian people, especially the youth who were the main participants, it is only the first step of many to reestablish a new Tunisian government with true democratic values and practices. The very young Tunisians who were so persistent in driving away Ben Ali are those who are still taking their roles as Tunisian citizens in order to continue the process toward democracy and the scheduled elections on July 24th. Prior to the revolution, Ben Ali’s ruling party was consistently elected to power, and his censorship of other political entities essentially prohibited the success of opposing campaigns. For this, the Tunisian population was neither educated about the political process nor did it have interest in staying informed about Ben Ali’s masquerade government. However, the revolution has opened the door to freedom in politics, and the developing political freedom has led to the creation of political parties that never had the opportunity to exist under Ben Ali’s rule. While these parties are frantically trying to prepare themselves in order to be viable candidates for the upcoming elections, young Tunisians are trying to educate themselves about democratic politics after a twenty-three year period of apathy due to the blatant abuse of democracy by Ben Ali’s regime

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

Recent advances on the taxonomy, systematics and epidemiology of Trichinella

Since Owen first described Trichinella as a human pathogen in 1835, the number of organisms comprising this genus has grown dramatically. Where it was once thought to be a monospecific group, this genus is now comprised of eight species and three additional genotypic variants that have yet to be taxonomically defined. Along with the growth in the genus and description of the parasites has come a concomitant increase in our understanding of the epidemiology and geographical distribution of these organisms. Recent expansion of the non-encapsulated group to include three species biologically defined by their unique host ranges encompassing mammals, birds and reptiles, has raised substantial questions as to the term, ‘Trichinella-free’ as it applies to geographical localities. A true appreciation of the adaptability of this genus to host and environmental selection factors, as well as its dissemination to the far reaches of the world can best be appreciated by reviewing what we know and what we hope to know about this ancient and elusive parasite. The review herein consolidates our current understanding of the taxonomy, epidemiology, and phylogeny of the genus Trichinella, and identifies areas where data are lacking and our knowledge requires additional clarification

A multiplex PCR assay for differentiating economically important gastrointestinal nematodes of cattle

A multiplex polymerase chain reaction (PCR) test was developed for identifying gastrointestinal (GI) nematodes that commonly infect cattle. This assay was developed using adult-derived genomic DNA and shown capable of discriminating parasite eggs from the feces of experimentally-infected animals at both the species and genus levels. Sequence data from internal (ITS) and external (ETS) transcribed spacers of the ribosomal DNA (rDNA) repeats as well as the 3′-end of the small subunit rDNA and 5′-end of the large subunit rDNA were used to generate five primer sets which, when used simultaneously in a multiplex PCR, produce a unique electrophoretic DNA banding pattern charac- terized by a single DNA fragment for Ostertagia ostertagi (257 bp), Haemonchus placei (176 bp),Oesophagostomum radiatum (329 bp), Trichostrongylus colubriformis (243 bp) and Cooperia on- cophora (151 bp). In a similar manner, the constructed primer sets amplified DNA from Ostertagia lyrata, Haemonchus contortus, Trichostrongylus axei, Cooperia surnabada and Cooperia punctata. With respect to H. contortus, a closely migrating doublet was generated suggesting size heterogene- ity in the ETS which is consistent with multiple rDNA repeat units within this species. PCR analyses using mixtures of monospecifically-purified nematode eggs indicated a sensitivity of less than 0.5 egg-DNA equivalent per species. Although, not designed as a quantitative technique, relative PCR signal intensities corresponded to relative egg burdens within the DNA samples from mixed species of eggs. Published by Elsevier Science B.V

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

Developments and hurdles in generating vaccines for controlling helminth parasites of grazing ruminants

As a direct consequence of rising drug resistance among common nematodes of grazing animals, efforts toward state-of-the- art vaccine development have clearly intensified in recent years, fuelled primarily by the advent of newer technologies in gene discovery, by advancements in antigen identification, characterisation and production. In this regard, it is appropriate to review progress that has been made in generating helminth vaccines and in particular, vaccines against common nematodes of production animals for consumption. In like manner, it is prudent to evaluate barriers that have hindered progress in the past and continue to present obstacles that must be solved when utilizing and depending on host immunity to attenuate parasitic infections