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We examined the immunotoxic effects of acute and subchronic exposures of goldfish to aged, fresh, and ozonated oil sands process-affected water (OSPW) using a flow-through exposure apparatus. We measured the expression of proinflammatory cytokine genes, the antimicrobial responses of primary macrophages isolated from OSPW-exposed fish, and the ability of the goldfish to control infection with a protozoan parasite, Trypanosoma carassii. After acute (1 week) exposure to aged OSPW, we observed upregulation in the expression of interferon gamma (IFN-γ), tumor necrosis factor alpha-2 (TNF-α2) in the kidney and spleen but not in gills of the fish. After subchronic (12 weeks) exposure to aged OSPW, we observed significant increases in mRNA levels of proinflammatory genes in the gill (IFN-γ, interleukin-1 beta 1 [IL1-β1], TNF-α2), kidney (IL1-β1, TNF-α2), and spleen (IL1-β1). An upregulation of immune gene expression in the gill and kidney (IFN-γ, IL1-β1, TNF-α2) and spleen (IL1-β1, TNF-α2) was observed after acute exposure of fish to diluted fresh OSPW. Following subchronic exposure to diluted fresh OSPW, we observed high mRNA levels of IL1-β1 in all tissues examined. However, there were significant decreases in the mRNA levels of IFN-γ and TNF-α2 in the kidney and spleen and gill and spleen (IL-12p35 and IL-12p40) of exposed fish. There were no changes in the expression of anti-inflammatory cytokine IL-10 after both acute and subchronic exposures to diluted fresh OSPW. In fish exposed to ozonated fresh OSPW, immune gene expression was similar to nonexposed control fish in all organs examined, with exception of IL1-β1. The ability of primary kidney macrophages to generate reactive oxygen and nitrogen intermediates was significantly reduced in fish exposed to fresh OSPW. The enhanced proinflammatory response after acute exposure to diluted fresh OSPW was confirmed by the parasite challenge experiments, where OSPW-exposed fish controlled the infection better than nonexposed fish.

Removal of the poly(A) tail is the first step in the degradation of many eukaryotic mRNAs. In metazoans and yeast, the Ccr4/Caf1/Not complex has the predominant deadenylase activity, while the Pan2/Pan3 complex may trim poly(A) tails to the correct size, or initiate deadenylation. In trypanosomes, turnover of several constitutively-expressed or long-lived mRNAs is not affected by depletion of the 5'-3' exoribonuclease XRNA, but is almost completely inhibited by depletion of the deadenylase CAF1. In contrast, two highly unstable mRNAs, encoding EP procyclin and a phosphoglycerate kinase, PGKB, accumulate when XRNA levels are reduced. We here show that degradation of EP mRNA was partially inhibited after CAF1 depletion. RNAi-targeting trypanosome PAN2 had a mild effect on global deadenylation, and on degradation of a few mRNAs including EP. By amplifying and sequencing degradation intermediates, we demonstrated that a reduction in XRNA had no effect on degradation of a stable mRNA encoding a ribosomal protein, but caused accumulation of EP mRNA fragments that had lost substantial portions of the 5' and 3' ends. The results support a model in which trypanosome mRNAs can be degraded by at least two different, partially independent, cytoplasmic degradation pathways attacking both ends of the mRNA.

Two Trypanosoma cruzi-derived cloning vectors, pTREX-n and pBs:CalB1/CUB01, were used to drive the expression of green fluorescent protein (GFP) and DsRed in Trypanosoma rangeli Tejera, 1920, and Trypanosoma cruzi Chagas, 1909, isolates, respectively. Regardless of the species, group, or strain, parasites harboring the transfected constructs as either episomes or stable chromosomal integrations showed high-level expression of fluorescent proteins. Tagged flagellates of both species were used to experimentally infect Rhodnius prolixus Stal, 1953. In infected bugs, single or mixed infections of T. cruzi and T. rangeli displayed the typical cycle of each species, with no apparent interspecies interactions. In addition, infection of kidney monkey cells (LLC-MK2) with GFP-T. cruzi showed that the parasite retained its fluorescent tag while carrying out its life cycle within cultured cells. The use of GFP-tagged parasites as a tool for biological studies in experimental hosts is discussed, as is the application of this method for copopulation studies of same-host parasites.

Trypomastigotes attached to the wall of the hypopharynx in tsetse flies infected with Trypanosoma vivax are believed to represent the true metacyclic stage of this trypanosome. Electron microscopy demonstrates that attachment is mediated by hemidesmosome-like junctions along the flagellar membrane and that none of the trypomastigotes, either attached or free in the hypopharynx lumen, possesses a surface coat comparable with that on the metacyclics of T. brucei and T. congolense and on the bloodstream stages of all salivarian trypanosomes. As the variable antigen of bloodstream and metacyclic T. brucei is located in the surface coat, the absence of the coat from metacyclic T. vivax suggests that the mechanism of antigenic variation in this species may be somewhat different from that of antigenic variation in T. brucei, and that vaccination of cattle against T. vivax may prove a simpler proposition than vaccination against T. brucei.

The inability to cultivate infective bloodstream forms of the African trypanosomes in cell-free media has complicated studies of the biology of trypanosomes and the pathogenesis of trypanosomiasis. We attempted to overcome this problem by subcutaneous implantation in mice of Millipore chambers that isolate trypanosomes from cells but permit diffusion of soluble substances across their membranes. Chambers were inoculated with 5 X 10(4) to 5 X 10(5) per ml Trypanosoma brucei, T. rhodesiense or T. gambiense; the trypanosomes multiplied rapidly, persisted for as long as five weeks, and remained infective, even when the original inocula were freed of donor cells by ion-exchange. The presence of anti-trypanosomal IgG and IgM in the sera and chambers of recipient mice proved that trypanosomal and mammalian products crossed the membranes. Chamber trypanosomes also expressed two important aspects of normal in vivo biological behaviour: (i) differentiation from long slender to short stumpy bloodstream forms and (ii) antigenic variation. Death of trypanosomes was associated with the presence of IgM antibody in the chambers. This model provides a system for study of an entire population of trypanosomes in an extravascular, cell-free environment.