Dante S. Zarlenga

The draft genome of the parasitic nematode Trichinella spiralis

Genome evolution studies for the phylum Nematoda have been limited by focusing on comparisons involving Caenorhabditis elegans. We report a draft genome sequence of Trichinella spiralis, a food-borne zoonotic parasite, which is the most common cause of human trichinellosis. This parasitic nematode is an extant member of a clade that diverged early in the evolution of the phylum, enabling identification of archetypical genes and molecular signatures exclusive to nematodes. We sequenced the 64-Mb nuclear genome, which is estimated to contain 15,808 protein-coding genes, at ∼35-fold coverage using whole-genome shotgun and hierarchal map–assisted sequencing. Comparative genome analyses support intrachromosomal rearrangements across the phylum, disproportionate numbers of protein family deaths over births in parasitic compared to a non-parasitic nematode and a preponderance of gene-loss and -gain events in nematodes relative to Drosophila melanogaster. This genome sequence and the identified pan-phylum characteristics will contribute to genome evolution studies of Nematoda as well as strategies to combat global parasites of humans, food animals and crops.

A multiplex PCR for unequivocal differentiation of all encapsulated and non-encapsulated genotypes of Trichinella

We have developed a single PCR test for the simple and unequivocal differentiation of all currently recognised genotypes of Trichilnella. Partial DNA sequence data were generated from internal transcribed spacers ITS1 and ITS2, and from the expansion segment V region of the ribosomal DNA repeat from five species of Trichinella and two additional genotypes, designated T5 and T6. Five different PCR primer sets were identified which, when used simultaneously in a multiplex PCR, produce a unique electrophoretic DNA banding pattern for each species and genotype including three distinct genotypes of Trichinella pseudospiralis. The banding patterns for each parasite genotype consist of no more than two well-defined DNA fragments, except isolates of T. pseudospiralis which generate multiple, closely migrating bands. The expansion segment V-derived primer set contributes at least one fragment to each genotypic pattern and, therefore, functions both as a means for differentiation as well as an internal control for the PCR. The reliability and reproducibility of each DNA banding pattern were verified using multiple geographical isolates of each Trichinella genotype. The technique was developed further to distinguish genotypes at the level of single muscle larvae using a nested, multiplex PCR, whereby the entire internal transcribed spacer region as well as the gap region of the expansion segment V of the large subunit ribosomal DNA are amplified concurrently in a first-round PCR using primer sets specific for each region, followed by the multiplex PCR for final diagnosis.

Cryptosporidium parvum infection in bovine neonates: dynamic clinical, parasitic and immunologic patterns

Twenty-six experimentally infected calves were monitored daily for oocyst excretion. All began excreting oocysts 3-6 days p.i. Most calves (n = 23) excreted oocysts for 6-9 days, with a daily range from 4 x 10(2) to 4.15 x 10(7) oocysts g(-1) of faeces. Over half the calves excreted peak numbers of oocysts 6-8 days p.i. Diarrhoea, observed intermittently beginning as early as day 3 p.i., lasted 4-16 days and varied greatly in severity from calf to calf. In a second study, nine of 18 calves were orally inoculated with 5 x 10(6) oocysts between birth and 2 days of age and nine remained uninfected. Monoclonal antibodies for cell surface markers indicated substantial increases in CD4+ and CD8+ T cells in the intraepithelial lymphocyte population of the ilea of infected calves at 7-9 days of age. RT-PCR demonstrated increases in mRNA for interleukin-12 and interferon-gamma that correlated with increases in both CD4+ and CD8 + intraepithelial lymphocyte cells. Increased mRNA for interleukin-12 and interferon-gamma from lamina propria lymphocytes correlated with increased numbers of CD8+ cells. No changes were found in interleukin-2, interleukin-4 or interleukin-10 mRNA levels. However, interleukin-15 mRNA, possibly from epithelial cells contaminating intraepithelial lymphocytes, was decreased in infected calves and had a negative correlation with increases in CD4+ and CD8+ cells. No differences were detected in mRNA levels for cytokines from lymph node lymphocytes.

Molecular taxonomy, phylogeny and biogeography of nematodes belonging to the Trichinella genus

Studying parasites of the genus Trichinella provides scientists of today many advantages. This is a group of zoonotic nematodes that circulates freely among wildlife hosts with one in particular, Trichinella spiralis that is exceptionally well adapted to domestic swine. Recent reports suggest that human infections from hunted animals are on the rise worldwide and numerous countries still experience problems with T. spiralis in their domestic food supplies. Trichinella is a genus whose members are easily propagated in the laboratories, have been used as models to investigate host-parasite relationships and parasitism among clade I organisms, and represent a poorly investigated link between the phylum Nematoda and other Metazoans. The importance of T. spiralis in better understanding the tree of life was so recognized that in 2004, its genome was carefully selected as one of only nine key non-mammalian organisms to be sequenced to completion. Since it was first discovered in 1835, this genus has expanded from being monospecific to eight species including four other genotypes of undetermined taxonomic rank. Inasmuch as discriminating morphological data have been scant, our understanding of the genus has been relegated to a compilation of molecular, biochemical and biological data. Herein, we provide a collection of information and up-to-date interpretations on the taxonomy, diagnostics, systematics, micro- and macroevolution, and the biogeographical and biohistorical reconstruction of the genus Trichinella.

New pieces of the Trichinella puzzle.

Contrary to our understanding of just a few decades ago, the genus Trichinella now consists of a complex assemblage of no less than nine different species and three additional genotypes whose taxonomic status remains in flux. New data and methodologies have allowed advancements in detection and differentiation at the population level which in turn have demonstrably advanced epidemiological, immunological and genetic investigations. In like manner, molecular and genetic studies have permitted us to hypothesise biohistorical events leading to the worldwide dissemination of this genus, and to begin crystalising the evolution of Trichinella on a macro scale. The identification of species in countries and continents otherwise considered Trichinella-free has raised questions regarding host adaptation and associations, and advanced important findings on the biogeographical histories of its members. Using past reviews as a backdrop, we have ventured to present an up-to-date assessment of the taxonomy, phylogenetic relationships and epidemiology of the genus Trichinella with additional insights on host species, survival strategies in nature and the shortcomings of our current understanding of the epidemiology of the genus. In addition, we have begun compiling information available to date on genomics, proteomics, transcriptomics and population studies of consequence in the hope we can build on this in years to come.

The systematics of the genus Trichinella with a key to species

The authors review the major biological, biochemical, and molecular characters that are used to distinguish the seven Trichinella species (T. spiralis, T. nativa, T. britovi, T. pseudospiralis, T. murrelli, T. nelsoni, T. papuae) and three genotypes whose taxonomic status is yet uncertain (T-6, T-8, T-9). A comparison of host specificity, morphology, reproductive abilities, nurse cell development and freeze resistance is presented, along with useful biochemical and molecular markers. Finally, this information is used to construct a diagnostic key for the species. A phylogenetic classification of the species is needed.

Expression and functional characterization of recombinant chicken interferon-gamma

A cDNA encoding chicken interferon-gamma (chIFN-gamma) was cloned from a CD4+ T-cell hybridoma by reverse transcription-polymerase chain reaction (RT-PCR) and expressed in Escherichia coli, COS- and CEC-32 fibroblast cell lines. In general, recombinant chicken IFN-gamma (rchIFN-gamma) expressed in the COS- and CEC-32 cell lines showed high bioactivity in vitro. The kinetics of IFN-gamma gene expression were examined in concanavalin A (Con A)-activated spleen lymphocytes by Northern blot and RT-PCR. IFN-gamma mRNA was detected as early as 30 min after Con A activation, reached peak expression at 2 h and then decreased starting at 4 h post Con A activation. A rabbit serum made to a synthetic peptide of IFN-gamma immunoprecipitated a 60 kDa E. coli maltose-binding fusion protein of recombinant IFN-gamma (MBP-IFN) and a 26-27 kDa secreted protein from COS cells and Con A-activated spleen cells. IFN-gamma inhibited vesicular stomatitis virus (VSV) mediated cytotoxicity of chicken embryonic fibroblast (CEF) cells and upregulated the expression of many macrophage cell surface antigens, including class I and class II major histocompatibility complex (MHC) proteins. These results show that chicken IFN-gamma possesses anti-viral activity and immunoregulates macrophage activities.

Characterization and detection of a newly described Asian taeniid using cloned ribosomal DNA fragments and sequence amplification by the polymerase chain reaction

DNA isolated from a newly described taeniid from Taiwan which shows adult characters indistinguishable from those of Taenia saginata was compared to DNA from T. saginata and nine other cestodes by restriction endonuclease digestion of genomic DNA and Southern blot analysis using 32P-labeled total cestode RNA and cloned ribosomal RNA gene fragments as probes. Hybridization patterns of Taiwan Taenia DNA revealed distinct variations from that of T. saginata and Taenia solium as well as all other cestode DNAs examined; however, similarities in restriction maps and sequence data between cloned ribosomal gene fragments from Taiwan Taenia (pTTr 3.1) and T. saginata (pTSgr 3.1 and PTSgr 2.4, respectively) suggest close evolutionary relatedness between these two taeniids. DNA sequence amplification from genomic DNA using oligonucleotide primers homologous to regions on both the 2.4- and 3.1-kb fragments was able to delineate between Taiwan Taenia and T. saginata by generating 1.0- and 0.29-kb fragments, respectively. Results demonstrated that Taiwan Taenia is not exclusive to Taiwan but exists in other parts of Eastern Asia and that adult morphology is insufficient for its detection in other locations. Results further support biological data indicating that Taiwan Taenia and T. saginata, although similar morphologically, are distinct genotypes.

VARIATIONS IN MICROSATELLITE SEQUENCES PROVIDE EVIDENCE FOR POPULATION DIFFERENCES AND MULTIPLE RIBOSOMAL GENE REPEATS WITHIN TRICHINELLA PSEUDOSPIRALIS

Enzymatic amplification of expansion segment 5 sequences within domain IV of the large subunit ribosomal DNA generated distinct results among geographical isolates of Trichinella pseudospiralis from Russia, North America, and Australia from both avian and mammalian hosts. Discrete, multiple DNA fragments ranging in approximate size from 285 to 360 bp were observed within and among each of the isolates tested. Polymerase chain reaction performed on individual adult parasites from each isolate resulted in multiple DNA fragments that were comparable to those generated from pooled genomic DNA. Sequence analysis of cloned, representative amplified fragments demonstrated that fragment length variation resulted primarily from the dinucleotide (TG)n and trinucleotide (TGC)n microsatellite repeats present within the expansion segment. Results are consistent with both population differences within the species as well as the presence of multiple alleles of the large subunit ribosomal RNA genes within individual parasites.

Molecular cloning and expression of an immunodominant 53-kDa excretory-secretory antigen from Trichinella spiralis muscle larvae

Abstract A Trichinella spiralis cDNA expression library was constructed in λgt11 from muscle larvae mRNA and immunologically screened to identify genes encoding previously described immunodiagnostic excretory-secretory (ES) antigens. Screening the library with T. spiralis infection serum from swine or rabbit antiserum to T. spiralis ES antigen identified one clone, designated TsA-12, that contains a cDNA transcript 539 bp in length and codes for an apparent 123-kDa β-galactosidase fusion protein that does not cross-react with Trichuris suis or Ascaris suum infection serum. Western blots of T. spiralis extracts and immunoperoxidase staining of tissue sections from muscle larvae using antibodies to purified TsA-12 demonstrate homology between TsA-12 and the 53 kDa diagnostic antigen from ES products (designated Ts.53) and localize the homologous native antigen to the stichocyte cells of the parasite. ELISA tests using TsA-12 as antigen, detected antibodies to T. spiralis in experimentally-infected mice as early as 14 days post-inoculation with maximum antibody titers being reached at 28 days post-inoculation. The TsA-12 dscDNA hybridizes to mRNA sequences expressed in both the muscle larvae and adult stages; however, concomitant expression of the native antigen is not observed within adult ES products. Southern blots of homologous and heterologous genomic DNAs probed with 32 P-labeled TsA-12 dscDNA fragments verify TsA-12 as a T. spiralis specific sequence that is present in multiple copies within the parasite genome.