L. H. Chappell

Human tumor necrosis factor α influences rainbow trout Oncorhynchus mykiss leucocyte responses

Abstract Human recombinant tumor necrosis factor α (rTNFα) and rainbow trout macrophage activating factor (postulated γ-interferon (γ-IFN) analogue) synergised to elevate the respiratory burst activity of trout macrophages. This elevated response may parallel phenomena described in mammals where TNFα and γ-IFN commonly synergise. Human rTNFα also synergised with mitogenic stimuli to heighten proliferation responses of trout head kidney leucocytes. These data are indicative of a conserved TNFα receptor on trout leucocytes (and possibly the TNFα molecule itself) and support the notion of an interactive cytokine network regulating immune responses in fish.

Cyclosporin A: antiparasite drug, modulator of the host-parasite relationship and immunosuppressant

Cyclosporin A (CsA), a cyclic undecapeptide with powerful properties of immunosuppression, acts on parasitic infections in laboratory animals in various ways. The outcome of drug administration in vivo varies with timing of treatment relative to infection, route of administration, dose and number of treatments applied. CsA is clearly antiparasitic against malaria, schistosomes, adult tapeworms, metacestodes and filarial nematodes. By contrast, it acts as an immunomodulator against trypanosomes and Giardia, by exacerbating infection; in the case of Leishmania spp. the drug acts variously. In some other infections CsA acts both as an antiparasite drug and as an immunosuppressant (Toxoplasma, avian coccidiosis and gastrointestinal nematodes). This range of activities is reviewed and possible modes of action discussed in the light of emerging data on in vitro drug activity and on putative receptor binding. The potential value of a non-immunosuppressive analogue of CsA in the control of parasitic infections of humans and domestic animals is considered but this paper lays particular stress on the seminal role of CsA as a laboratory tool.

Studies on the infectivity of Diplostomum spathaceum in rainbow trout (Oncorhynchus mykiss).

The infectivity of Diplostomum spathaceum (Digenea: Trematoda) cercariae to rainbow trout and the efficacy of the diplostomule migration to the lens following different routes of administration was examined. The optimum age of infectivity for cercariae was between 0-5 h after liberation from the snail and for intraperitoneally injected diplostomules, 5 h post-transformation in vitro through fish skin. After exposure of the entire fish body or head to cercariae, metacercariae first appeared in the lens at 5 h and their numbers gradually increased until 22 h. Following exposure of the tail region of rainbow trout to cercariae, metacercariae first appeared in the lens at 14 h. Significantly more metacercariae established in the lens of fish following exposure of the fish head compared with the tail region; 40% of penetrating cercariae reached the lens of fish following exposure of the head or entire body, 20% of cercariae or diplostomules injected either intraperitoneally, intramuscularly or intracardially reached the lens while only 5% of cercariae established as metacercariae following exposure of the tail region.

The antiparasite effects of cyclosporin A: possible drug targets and clinical applications

1. The immunosuppressive drug cyclosporin A, and some of its nonimmunosuppressive derivatives, are potent inhibitors of a range of parasites of humans. 2. Cyclosporin A and the structurally unrelated immunosuppressant FK506 are known to act on T-lymphocytes as complexes with their binding proteins, cyclophilins and FKBPs, respectively. 3. Cyclophilins and FKBPs have been structurally identified in a number of parasites and, in some instances, are believed to play roles in the antiparasitic actions of these drugs. 4. Nonimmunosuppressive cyclosporins and FK506 derivatives may have clinical potential in certain parasitic diseases, especially malaria and schistosomiasis, and identification of the targets of these drugs in parasites may lead to development of novel chemotherapeutic agents.

Chitinase in female Onchocerca gibsoni and its inhibition by allosamidin.

Chitinase activity has been detected in female worms of Onchocerca gibsoni. With 3,4-dinitrophenyl-tetra-N-acetylchitotetraoside as substrate 50% of maximum activity was achieved at about 25 microM substrate, with inhibition above 50 microM substrate. The antibiotic allosamidin very strongly inhibited the chitinase activity, 50% inhibition being achieved by 200 pM allosamidin in the presence of 45 microM substrate.

Phylogeny of Onchocerca volvulus and related species deduced from rRNA sequence comparisons

A rapid, direct method for determining the partial nucleotide sequence of large subunit ribosomal RNA was adapted and applied to a group of helminth parasites. Small samples of total, unfractionated cellular RNA isolated from each organism were analysed and the nucleotide sequences of equivalent portions of the large subunit ribosomal RNA compared. The data obtained were used to construct a phylogenetic tree showing the evolutionary relationships within this group of organisms.

Chitinolytic activities in Heligmosomoides polygyrus and their role in egg hatching

The occurrence of chitin in the eggshell of Heligmosomoides polygyrus has been determined by histochemical and biochemical techniques. Approximately 5% of the egg dry weight was chitin. Staining with Calcofluor white showed the chitin in the eggshell to be more accessible to the stain after hatching or rupturing of the eggshell. Chitinolytic activity has been detected using fluorescent substrates in extracts of adult males (at low levels), females and eggs. Enzyme activity in situ, within the developing larvae, was visualised with the same substrates. It was localized in discrete granules about 1 micron in diameter which occurred as groups in areas of about 5 microns in diameter, in the posterior third of the larvae. The chitinolytic activity in the eggs increased with the age of the egg and was released into the medium when the eggs hatched. The chitinase activities were very sensitive to inhibition by allosamidin, a specific chitinase inhibitor, with an IC50 for the crude egg extract of 2.2 nM. However, treatment of eggs with 250 microM allosamidin resulted in a slowing but not cessation of egg hatching.

Chitin in egg shells of Onchocerca gibsoni and Onchocerca volvulus

Chemical analysis of adult females of Onchocerca gibsoni gave estimated chitin contents of 200-500 micrograms (g dry weight)-1. Egg shells from both O. gibsoni and Onchocerca volvulus stained with Calcofluor white and with fluorescent wheat germ agglutinin as shown by fluorescent light microscopy, and bound gold-labelled wheat germ agglutinin as shown by electron microscopy, under conditions specific for chitin. The egg shells appeared as single electron dense layers from 50 to 85 nm in thickness. Purified chitinase digested these egg shells, leaving coiled microfilariae unattacked. We conclude that chitin is a major component of the egg shells.

The epidemiology of diplostomiasis in farmed rainbow trout from north-east Scotland

An epidemiological survey of the distribution and abundance of Diplostomum spathaceum in farmed rainbow trout (Salmo gairdneri) from Mill of Cantray trout farm (Nairn, Morayshire) involved monthly sampling of fish over a 3-year period. Diplostomum metacercariae were present in both the lens and aqueous humour of infected fish, and these have been treated separately throughout the study. The infection period was normally between May and September each year and transmission of the parasite from snail to fish did not occur at temperatures below 10 degrees C. The prevalence and abundance of both lens and humour metacercariae reached a maximum in September. The cleansing and application of a molluscicide (copper sulphate) to the raceways in spring resulted in a 56% reduction in the numbers of metacercariae infecting trout during the following summer. However, no further improvement in parasite control was recorded when the treatment was repeated in the following year. Experiments using caged fish indicated that diplostomiasis was confined to certain areas of the farm only and that the infection rate of rainbow trout with D. spathaceum cercariae was correlated (P less than 0.01) with water temperature. The results of the study indicated that it is possible by regular cleaning and use of molluscicides to keep the intensity of diplostomiasis at such a level that rainbow trout do not become severely affected. However, as with other parasitic diseases, a combination of control methods will probably be required to eradicate the disease completely from trout farms.

Cyclosporin A: Immune suppressant and antiparasitic agent

In 1976, following the first description of. its biological properties I the fungal metabolite cydosporin A (CsA) was hailed as the first of a new generation of remarkable immunosuppressive agents (see Box I). This accolade was due to the unique, discriminatory action of CsA on the activation of T helper/inducer (Tw,)lymphocytes, without the undesirable side effects associated with conventional immune suppressants, such as azathioprine and steroids. Since then, CsA has become established as the agent of choice in the control of human kidney and heart allograft rejection, greatly improving the results of organ transplant centres worldwide. Moreover, the advent of CsA has contributed significantly to the improved outcome of human liver transplantation and has led to a dramatic increase in the number of human pancreas transplants performed. There is currently considerable interest in the prospective value of CsA in the treatment of autoimmune disorders such as systemic lupus erythematosus. Due to its usefulness as a probe for dissecting the role ofT-cells in immunological responses, CsA has attracted the attention of experimental parasitologists. It came as some surprise however, when in 1981 the drug was found to have antiparasitic effects against murine schistosomiasis 2 and malaria 3. More recently, the spectrum of an~parasitic actJvities of CsA has been found to include toxoplasmosis 4 and filariasis s, although trypanosomiasis 6 and giardiasis 7 appear to be unaffected. Bueding et oL 2 suggested that specific inhibition of the haemoglobinase activity of female worms, following administration of CsA to the host, could account for the antischistosomal effect. However, the drug did not inhibit haemoglobinase activities of schistosomes in vitro, and a direct toxic effect of CsA against schistosomula has not been demonstrated in vitro even in the presence of serum from infected mice. More recently, Bout et oi. 8 and Nilsson et eL 9 have confirmed the protective effect of CsA against schistosomiasis mansoni and corroborated the observation that the drug is most effective against immature stages of the worms during the early stages of infection. In mice treated with CsA (30 mg/kg/day) from day I (infection on day 0) to day 3, only 10-30% of schis-