John F. Ryley

Azole resistance in Candida albicans

Two isolates of Candida albicans from chronic mucocutaneous candidosis patients who initially responded to ketoconazole treatment but who later relapsed, have shown an abnormal response to ketoconazole in four out of five systems in vitro and in three animal models of vaginal or systemic infection. They have also shown abnormal resistance to inhibition of ergosterol biosynthesis in whole cells, but not in cell-free systems, and to inhibition of amino acid uptake. We conclude that the behaviour of the isolates is consistent with the development of drug resistance to ketoconazole. In all systems the two isolates have shown cross-resistance to the triazole antifungal ICI 153,066. In addition they fail to take up radiolabelled ICI 153,066--in contrast to normal isolates--indicating that resistance is due to changes in the properties of the cell membrane rather than internal enzymology.

A Comparison of Phospholipase Activity, Cellular Adherence and Pathogenicity of Yeasts

Phospholipase A and lysophospholipase activities were measured in the culture fluid and in the blastospores of Candida albicans. When phospholipase activity was measured in six yeasts (four strains of C. albicans and a single strain each of Candida parapsilosis and Saccharomyces cerevisiae) a correlation was found between this activity and two potential parameters of pathogenicity. The C. albicans isolates which adhered most strongly to buccal epithelial cells and were most pathogenic in mice had the highest phospholipase activities. Non-pathogenic yeasts, including C. albicans isolates which did not adhere and did not kill mice, had lower phospholipase activities.

Chemotherapy of Chicken Coccidiosis

Publisher Summary This chapter discusses the chemotherapy of chicken coccidiosis. Coccidiosis of the chicken can be caused by any one of nine species of parasite, either alone or in combination. Only six of the species— Eimeria acervulina , Eimeria brunetti , Eimeria maxima , Eimeria mivati , Eimeria necatrix , and Eimeria tenella are thought to be of economic significance. E. necatrix and E. tenella can give rise to spectacular outbreaks of disease, with blood-stained litter and appreciable mortality, whereas other species are more insidious in their attack. The greater part of the coccidial developmental cycle takes place in the chicken, and most attempts at control have involved the use of preventive or therapeutic medication in the host. The source of infection however, is the omnipresent oocyst, passed out at the end of the life cycle in the bird, and subsequently undergoing the process of sporulation on the ground before becoming infective to a new host. The chapter also discusses the modes of drug action and drawing attention to the possibility of inhibiting development of coccidia without actually killing the parasite. Drugs that have activity against coccidia are usually referred to as coccidiostats.

Methods in coccidiosis research: separation of oocysts from faeces

Factors which may be important in the large-scale extraction of coccidial oocysts from faeces ha.ve been investigated with Eimeria tenella. Age of bird, inoculum, feeding status at the time of inoculation, period of collection, feeding status during collection, collection medium, homogenization and sieving, flotation, washing, sporulation and further purification have all been considered. The aim has been to establish a method to produce the maximum number of oocysts of a required degree of purity and viability, with the expenditure of the minimum amount of physical effort, time, animals and chemicals. In our method, groups of chickens 3-4 weeks of age are inoculated with 5000 oocysts of E. tenella and food is supplied ad lib. Over the period 5-8 days after inoculation, faeces are collected in trays containing 2% (w/v) potassium dichromate solution, while food intake is restricted. The faecal material is homogenized, passed once through 40 and 100 mesh sieves, centrifuged and the oocysts recovered from the sediment by 3 flotations in saturated salt solution. Following washing, oocysts are sporulated by forced aeration at 30 degrees C and may be further purified by hypochlorite treatment, or passage in 5% Tween 80 solution through a glass bead column followed by sucrose density gradient centrifugation. Routine passages along these lines over a 5 year period have given a recovery of 46% of the oocysts excreted by over 7000 birds.

Experimental approaches to antifungal chemotherapy.

Publisher Summary This chapter discusses the experimental approaches to antifungal chemotherapy. Fungal diseases in general occur in all parts of the world and affect all ages, though a number of particular species are restricted geographically in their incidence. It goes without saying that a systemic fungal infection will require systemic treatment. When however the fungus is confined to the skin or mucosal membranes, then the possibility exists of applying a topical treatment to the affected area, or alternatively treating the infection systemically, getting the parasite from “behind.” With a topical treatment the patient may feel he is doing something positive by applying the medication to the lesion whereas an oral treatment may give the feeling of irrelevance. On the other hand there would seem little point in applying a messy treatment to an already messy lesion if an alternative oral treatment were available.

Laboratory studies with some older anticoccidials

Features of the anticoccidial activity of nicarbazin, amprolium, zoalene, sulphadimidine, diaveridine, Darvisul, spiramycin, chloramphenicol and oxytetracycline have been re-investigated both in vivo and in cell culture using Eimeria tenella. Of the drugs studied, only spiramycin was appreciably coccidiocidal, although nicarbazin and amprolium showed possibly slower coccidiocidal activity. In order to show activity against a particular stage in the life-cycle, higher concentrations of drug than those usually recommended for field usage had in most cases to be used. Under these conditions, parasites were usually inhibited as multinucleate 1st generation schizonts. With delayed medication, effects against 2nd generation parasites were in most cases found, and in many cases, although the parasites never matured to give viable merozoites, the large degenerating forms produced were able to cause extensive tissue destruction and haemorrhage. Methodology in this type of study is discussed in relation to more active and more recent anticoccidials, and some further experiments with robenidine reported.

Ultrastructural studies on the sporozoite of Eimeria tenella

The structure of the sporozoite of Eimeria tenella has been studied with the aid of the electron microscope, using both sectioned fixed material and negatively stained or shadowed whole cells. An apical ring gives rise to a series of 24 fibrils which extend posteriorly under a double bilamellar membrane. Within the apical ring lies a conoid with spirally arranged tubules and an apical pore; under some conditions at least this conoid can be pushed forward beyond the apical ring. Originating within the conoid are a group of club-shaped organelles which extend backwards into the cell to lie among an extensive regular array of micronemes. The nucleus lies between two apparently structureless paranuclear bodies, and scattered throughout the remaining cytoplasm are mitochondria with tubular cristae, oval structures containing amylopectin, occasional lipid droplets, and vesicles containing nondescript material possibly undergoing digestion and which may have a possible connexion with a lateral micropyle. I am indebted to Mrs Muriel Bentley for much technical collaboration and to Mrs McLaren for making her manuscript available before publication.

Studies on the Mode of Action of Quinolone and Pyridone Coccidiostats

The primary action of the anticoccidial drugs methyl benzoquate, buquinolate, and meticlorpindol is to prevent the growth of, rather than to kill, coccidia. In the case of Eimeria tenella in the chick, only the sporozoite when it has first invaded the host cell is susceptible to this action, but this is due not to lack of susceptibility of later stages, but rather to failure of the drug to penetrate deep into the gut wall. Later stages of E. brunetti are susceptible to methyl benzoquate given in the food as are later stages of E. tenella if the drug is given intravenously or the experiments are carried out in the chick embryo. Methyl benzoquate given intravenously in the chick forms a depot, mainly in the lung, which is able to protect against lethal coccidial challenges for several weeks. No success has been achieved in explaining the mode of action of the drugs at a molecular level, although the observations would be consistent with an inhibition of nucleic acid synthesis. The earliest compounds found to have marked anticoccidial activity were the sulphonamides, and as with bacteria, it has been shown that these act as competitive inhibitors of p-aminobenzoic acid (Horton-Smith and Boyland, 1946; Waletzky and Hughes, 1946). During studies involving treatment with varying concentrations of sulphadimidine over restricted parts of the life cycle of Eimeria tenella, it was shown (Kendall and McCullough, 1952; Davies and Kendall, 1954a, b) that the stages of the parasite present 72 to 96 hr after infection were most sensitive to the drug, whereas those present during the first 24 hr of the infection were only influenced by very high concentrations of sulphonamide. Moreover, although second generation schizonts appeared to be killed by sulphadimidine, early treatment of infections led only to suppression of the parasite, which was able to continue its development if the drug were later withdrawn. Potentiation of sulphonamides with substances such as pyrimethamine or diaveridine rely on the ability of the potentiator to interfere with the folic acid pathway in the parasite (Joyner and Kenldall, 1956). Amprolium was developed as a coccidiostat during a program of work on thiamine antagonists, and its anticoccidial activity can be annulled by thiamine in the chick (Rogers et al., 1960; Rogers, 1962) or in the chick embryo (Ryley, 1968). The present studies were undertaken in an attempt to throw some light on the mode of action of Received for publication 28 July 1967. the newly introduced quinolone coccidiostats methyl benzoquate (I.C.I. 55,052) (Bowie et al., 1967; Ryley, 1967) and buquinolate (Spencer et al., 1966) and the pyridone, meticlorpindol (Stock et al., 1967).

Activity of ICI 195,739—a Novel, Orally Active Bistriazole—in Rodent Models of Fungal and Protozoal Infections

ICI 195,739 shows superior potency to other azoles in eliminating vaginal candidosis or dermatophyte infections in animal models of infection by both oral dosing and topical application; effective doses are in the range of 0.5-5.0 mg/kg/day or 0.01-0.30% in a topical formulation. ICI 195,739 is likewise effective in models of systemic fungal infection; 1, 10, 25 mg/kg/day will protect animals given a lethal inoculum of C. albicans, C. neoformans, or A. fumigatus, respectively, as long as dosing is continued, showing activity in this respect superior to that of other azoles tested. ICI 195,739 will suppress infections in mice with T. cruzi and prevent mortality with five daily doses of 1 mg/kg; cure rather than suppression of patent infections has been achieved with 35 daily doses of 10 mg/kg.

Quantification of vaginal Candida albicans infections in rodents

Estradiol-treated mice and estradiol-treated ovariectomized rats support vaginal infections with Candida albicans for several months; low-grade uterine infections occur in around half the animals. A comparison has been made and the relative advantages discussed of quantifying these infections by sampling with a wire loop and plating on BiGGY agar, sampling by vaginal washing, or removal and homogenization of the vagina followed by dilution and plate counting.