The spicules of male parasitic nematodes are key morphological features, which vary between species in shape and length and are used often for species identification. However, little is known about spicules and particularly if/how their length varies during growth. We first assessed the degree of variation in spicule length of male Heligmosomoides bakeri 21 days post infection (PI), and then in two follow-up experiments measured spicule lengths at half daily/daily intervals between days 6 and 14 PI. Mean spicule length in 21-day worms was 0.518 mm with a range of 94 μm, and variation between the two spicules of individual worms from 2 to 32 μm. Spicules were first detectable on day 6-6.5, after which their lengths increased until day 7 PI (mean = 0.61 and 0.59). This was followed by significant contraction, initially relatively quickly over the following 48 h and then more slowly over a longer period, stabilizing by days 10-14, with only minor further reduction in length. We conclude that the length of spicules varies significantly over the first few days after they have formed, and, consequently, the age of worms is an important factor for consideration when spicule lengths are measured for experimental/diagnostic or taxonomical purposes.
Abstract Nematode spicules vary in shape and size even between closely related species and, therefore, constitute key characters in nematode taxonomy for distinguishing between species. Spicules are seldom measured on fresh specimens, but rather at some time after extraction from culled hosts and after a period of preservation of the worms in chemical fixatives or by freezing. We carried out two experiments to assess the effects of freezing in Hanks’ balanced salt solution, 70% or 80% ethanol and 10% formalin (both of the latter at room temperature and after storage at −80°C) on spicule length of Heligmosomoides bakeri at two time intervals after extraction from mice (Experiment 1, one and four weeks; Experiment 2, one and four months). In Experiment 1, no significant differences were detected, although there was some variation between treatments and over time. In Experiment 2, spicule length varied significantly between treatments and over time, the greatest shrinkage being in 80% ethanol and the least in 10% formalin. However, overall variation in spicule length was very limited, accounting for no more than 5.03% change in length over time and 4.95% between treatments at any of the periods of assessment. Therefore, while whole nematodes can shrivel and shrink in preservatives, making many measurements unreliable, our data indicated that spicule lengths are very little changed by preservation techniques over time, and so spicule length remains as a reliable taxonomic character.
Helminth parasites promote the development of antigen-specific anergy and regulation which often limits pathology but allows parasite survival. Parasite effectors mediating this are the subject of intense study as they may be useful as future vaccine targets. Fasciola hepatica possesses a TGF-like molecule (FhTLM), part of a larger TGF family, which play an important role in regulating parasite development and modulating host immune responses. We hypothesized that F. hepatica may utilise FhTLM to down modulate host immune responses via induction of regulatory. Consequently, in this study, the immuno-modulatory properties of FhTLM with regards to its effect on T-cell mediated immunosuppression and modulation of host immune cells and signals to benefit parasite survival were studied.
The in vitro effect of FhTLM on induction of FoxP3 T-regs cells and anergic markers, PD-1, PD-L1 and CTLA-4, was studied in murine splenic CD4 T-cells. FhTLM failed to upregulate FoxP3 T-reg cells and all investigated anergic T-cell markers. However, production of IL-10, a key immunomodulatory cytokine was significantly enhanced in response to FhTLM stimulation. The interaction of FhTLM with naive CD4 T-cells also promoted the development of inflammatory responses evidenced by enhanced production of pro-inflammatory cytokines and chemokines (TNF-α, IL-12 P70, IL-1β, IP-10, TARC, MIP-1β, IL-1ra, RANTES and CXCL12). The chemokines induced by FhTLM were biologically functional as supernatants from FhTLM stimulated cells promoted in vitro migration of both activated CD4 T-cells and eosinophils. Lastly, immune signals generated from CD4 T-cells, eosinophils and monocytes failed to support enhanced survival of newly excysted juvenile (NEJs) flukes and this response was not affected by the presence FhTLM. Whilst FhTLM did not directly modulate immune cells to promote parasite survival, induction of pro-inflammatory responses as evidenced by production of cytokines; IL-6, TNF-α, RANTES, MCP-1, Lipocalin -2, Endocan, Pentraxin-3 and ICAM-1 by LPS or IL-4-stimulated monocytes was suppressed or inhibited in the presence of FhTLM.
Taken together, our data highlight an indirect mechanism used by the parasite to benefit parasite persistence and also implicate a role for IL-10 in suppression of protective host immune response to F. hepatica infection. In addition, our data also indicates that FhTLM has an anti-inflammatory potential and may have implications in the treatment of allergic or pro-inflammatory diseases.
Abstract Nematode spicules play a vital role in the reproductive activity of species that possess them. Our primary objective was to compare the lengths of spicules of the laboratory mouse ( Mus musculus ) – maintained isolate H. bakeri – with those of H. polygyrus from naturally infected wood mice ( Apodemus sylvaticus ). On a more limited scale, we also included H. glareoli from bank voles ( Myodes glareolus ), a species reputed to possess longer spicules than either of the 2 former species. In total, we measured 1264 spicules ( H. bakeri , n = 614; H. polygyrus n = 582; and H. glareoli, n = 68). There was a highly significant difference between the spicule lengths of the Nottingham-maintained H. bakeri (mean = 0.518 mm) and H. polygyrus (0.598 mm) from 11 different localities across the British Isles. A comparison of the spicules of H. bakeri maintained in 4 different laboratories in 3 continents revealed a range in the mean values from 0.518 to 0.540 mm, while those of worms from Australian wild house mice were shorter (0.480 mm). Mean values for H. polygyrus from wood mice from the British Isles ranged from 0.564 to 0.635 mm, although isolates of this species from Norway had longer spicules (0.670 mm). In agreement with the literature, the spicules of H. glareoli were considerably longer (1.098 mm). Since spicules play a vital role in the reproduction of nematode species that possess them, the difference in spicule lengths between H. bakeri and H. polygyrus adds to the growing evidence that these 2 are quite distinct species and likely reproductively isolated.
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