Search

Genome assemblies can form the basis of comparative analyses fostering insight into the evolutionary genetics of a parasite's pathogenicity, host–pathogen interactions, environmental constraints and invasion biology; however, the length and complexity of many parasite genomes has hampered the development of well-resolved assemblies. In order to improve Trichinella genome assemblies, the genome of the sylvatic encapsulated species Trichinella murrelli was sequenced using third-generation, long-read technology and, using syntenic comparisons, scaffolded to a reference genome assembly of Trichinella spiralis, markedly improving both. A high-quality draft assembly for T. murrelli was achieved that totalled 63·2 Mbp, half of which was condensed into 26 contigs each longer than 571 000 bp. When compared with previous assemblies for parasites in the genus, ours required 10-fold fewer contigs, which were five times longer, on average. Better assembly across repetitive regions also enabled resolution of 8 Mbp of previously indeterminate sequence. Furthermore, syntenic comparisons identified widespread scaffold misassemblies in the T. spiralis reference genome. The two new assemblies, organized for the first time into three chromosomal scaffolds, will be valuable resources for future studies linking phenotypic traits within each species to their underlying genetic bases. [ABSTRACT FROM PUBLISHER]

ABSTRACTParelaphostrongylus tenuisis a parasitic nematode that causes a debilitating neurologic disease in many North American cervids and domestic livestock species. We produced a PCR-based cDNA library from infective larvae (L3) in order to identify molecules that mediate parasitism. A dominant 1,250-bp amplicon encoded a homologue of cathepsin B cysteine proteases. The sequence incorporated a C29G substitution in the putative active site. Antibodies generated against a recombinant form detected the native protein (PtCPR-1) in Western blot assays of L3, but not adult worm, extracts. Immunohistochemical methods revealed that PtCPR-1 synthesis was restricted to larval stages within the snail intermediate host (Triodopsissp.), beginning as early as 2 days postinfection (dpi) of snails. The protein was present in the intestine and luminal contents and was lost from larvae over time. Concurrent studies showed that larvae induced an immune response in snails beginning at 1 dpi. Layers of hemocytes encapsulated larvae immediately after infection, and granuloma-like structures formed around parasites in chronic infections. Loss of PtCPR-1 from L3 and its accumulation in host tissues coincided with degeneration of granuloma architecture 90 to 105 dpi. Fully developed L3 emerged from the snail at this time. Our data implicate PtCPR-1 in larval development and possibly in the emergence of P. tenuisfrom the intermediate host. Emerged L3 survived desiccation and cold stress, suggesting that they could remain infectious in the environment. Molecules promoting emergence would facilitate dispersal of L3 and increase the likelihood of transmission to definitive hosts.

Studies in mice and humans indicate that membrane CD14 (mCD14) on the surface of monocytes, macrophages and polymorphonuclear neutrophils (PMN) mediate activation of these cells by lipopolysaccharide (LPS). Soluble CD14 (sCD14), in the circulation, binds to LPS and blocks LPS binding to mCD14. The role of bovine CD14 in cellular activation by LPS is undefined. Changes in CD18 expression on PMN and steady state levels of mRNA for tumor necrosis factor-$\alpha$ (TNF-$\alpha$), interlukin-6 (IL-6) and IL-8, sensitive markers for activation of leukocytes by LPS, were used to measure functional activity of recombinant bovine sCD14 (rbosCD14). Whole blood (n = 3 cows) treated with LPS alone caused CD18 expression on PMN to increase by 12% ($P < 0.02$), whereas pre-incubation of LPS with 10 or 100 $\mu$g/mL of rbosCD14 completely inhibited increase in CD18 expression. After treating whole blood with LPS at concentrations of 1, 100 or 10$^{4}$ ng/mL for 2 h, level of mRNA for TNF-$\alpha$, IL-6 and IL-8 in leukocytes and concentration of TNF-$\alpha$ in plasma increased. However, pre-incubation of LPS with rbosCD14 inhibited the increase in TNF-a mRNA, but not the increase in IL-6 and IL-8 mRNA. Excess amount of anti-human CD14 monoclonal antibody (MAB) also inhibited LPS-induced increase in TNF-$\alpha$ mRNA. Preincubation of LPS with rbosCD14, or rbosCD14 plus MAB did not affect LPS-induced increase in TNF-$\alpha$ in plasma. Collectively, results indicate that rbosCD14 inhibit LPS-induced increase in CD18 expression and TNF-$\alpha$ mRNA. However, secretion of TNF-$\alpha$ was not inhibited by pre-incubation of LPS with rbosCD14. The TNF-$\alpha$ in plasma may partially induce transcription of IL-6 and IL-8, which contribute to the CD14-independent increase in level of mRNA for IL-6 and IL 8.

Delineation of the genus Trichinellainto a more complex group of parasites has substantially motivated investigators to better identify and characterize the species and genotypes that form the basis of their investigations. Because of the cosmopolitan geographical distribution and broad host range that typify this genus, assigning unique biological, immunological and biochemical characters to each species and genotype has been essential for researchers to further advance this field. Numerous groups have developed simple methods to differentiate the genotypes, and by so doing, have generated diagnostic keys that accurately reflect the distinct differences among parasites of this group. Throughout the years, many methods have been used to accomplish this task, beginning with isoenzyme analyses and the use of repetitive DNA probes, to employing the polymerase chain reaction (PCR) and more state-of-the-art technologies. This review article summarizes the development of these methods with emphasis on molecular techniques and the ultimate goal of providing a simple, rapid and reproducible test to differentiate Trichinellaparasites at the highest level of sensitivity, i.e. single parasite.

Interleukin-18 (IL-18) is a recently described cytokine that enhances interferon-γ (IFN-γ) production, either independently or synergistically with IL-12. These properties identify IL-18 as an immunoregulatory cytokine that may be pivotal in host defense against intracellular pathogens. We have isolated and sequenced a cDNA encoding bovine IL-18. The open reading frame (ORF) is 582 bp in length, encoding a predicted 192 amino acid (aa) precursor protein. Multiple sequence alignment demonstrated that bovine IL-18 has 65% and 78% identity with the predicted amino acid sequences of murine and human IL-18, respectively. IL-18 mRNA was constitutively present in bovine peripheral blood monocyte-derived macrophages (MDM), with no upregulation on stimulation with lipopolysaccharide (LPS). IL-18 transcripts were weakly detected in B lymphocytes but inducible in the B cell line BL-3. Human recombinant IL-18 (rHuIL-18) induced IFN-γ production by PHA-stimulated peripheral blood mononuclear cells (PBMC), which was potentiated by rHuIL-12. Further, rHuIL-12 and rHuIL-18 enhanced proliferation of untreated PBMC. Antigen-specific T cell lines demonstrated IL-18-dependent enhancement of IFN-γ production, indicating that bovine T cells are one of the leukocyte subsets that respond to IL-18. Analysis of IL-18 expression and its ability to induce IFN-γ production by bovine lymphocytes are important considerations for understanding mechanisms of protective immunity and designing vaccines for intracellular pathogens.

There has been a decline in the number of human trichinellosis cases associated with consumption of commercial pork in the United States, while the relative importance of trichinellosis from game meats has increased. An investigation of an outbreak of trichinellosis in Idaho occurring after consumption of improperly prepared cougar jerky is described. Ten cases of trichinellosis were identified among 15 persons who ate the implicated meat. Viable Trichinella larvae were recovered from frozen cougar tissue. Polymerase chain reaction on parasite DNA yielded results consistent with genotypes T. nativa and Trichinella type T6. This report of cougar meat as a source of human trichinellosis and the finding of freeze-resistant Trichinella organisms in wildlife in Idaho extends the range of this genotype. Consumers of game need to cook the meat thoroughly, since even frozen meat may harbor viable Trichinella that can cause illness.

Circular dichroism was used to examine alterations in the secondary structure of poly(dA-dT)·poly(dA-dT) upon binding polymer X, a polycationic CD probe for aspects of DNA structure. Stable complex formation is evidenced by increasing Tm and the appearance of large extrinsic bands in the > 300 nm, region which increase proportionally with r (ratio of polymer charge to DNA phosphate), in the range 0.0 to 0.32. At relatively low values of r (.32), CD spectra of the poly(dA-dT)·poly(dA-dT)-polymer X complex show a gradual non-cooperative inversion in the long wavelength portion (275 nm) of the intrinsic band in low salt solutions suggesting structural and conformational flexibility in poly(dA-dT)·poly(dA-dT) and further implicating polymer X as a potential probe for variations in DNA secondary structure. The dinucleotide repeat configuration of poly(dA dT)·poly(dA-dT) is presumed to play a role in the observed intrinsic CD changes. NMR data support an “alternating B” conformation for the complex.