George L. Stewart

Characterization and pathogenic potential of a soil isolate and an ocular isolate of Acanthamoeba castellanii in relation to Acanthamoeba keratitis

Acanthamoeba castellanii, one isolate from the eye and one from the soil, were compared on the basis of: (a) pathogenic potential; (b) plasminogen activator activity; (c) chemotactic activity; (d) cytopathic effects; (e) collagenolytic activity; (f) binding ability to contact lenses; and (g) and binding ability to corneal buttons. The ocular isolate of A. castellanii was found to be pathogenic based on its ability to produce corneal infections in Chinese hamsters. By contrast, the soil isolate produced only mild lesions in a single Chinese hamster. Amoebae from the ocular isolate bound to corneal epithelium in greater numbers than the soil isolate counterparts. Moreover, ocular isolate organisms displayed plasminogen activator activity that was not detected in cultures from soil isolates of A. castellanii. Although neither the soil isolate nor the ocular isolate amoebae responded chemotactically to epithelial or stromal components, the ocular isolate displayed a curious and reproducible positive chemotactic response to endothelial extracts. Both A. castellanii isolates produced cytopathic effects on pig corneal epithelium, however the cytotoxicity from the ocular isolate was significantly greater than that of the soil isolate. The results indicate that the pathogenic potential of A. castellanii is correlated with the parasites capacity to bind to corneal epithelium, respond chemotactically to corneal endothelial extracts, elaborate plasminogen activators, and produce cytopathic effects on corneal epithelium.

Chemotactic response of macrophages to Acanthamoeba castellanii antigen and antibody-dependent macrophage-mediated killing of the parasite

The chemotactic potential of antigens of Acanthamoeba castellanii for macrophages and the ability of naive and immune rat peritoneal macrophages to kill A. castellanii in vitro were assessed. The amoebolytic capacity of immune rat serum and complement was also examined. No parasite was killed in the presence of heat-inactivated naive rat serum. Low numbers of parasites were lysed in the presence of heat-inactivated immune rat serum, whereas significantly greater numbers of parasites were lysed in the presence of nonheat-inactivated naive and immune rat serum. Macrophages from naive rats were capable of lysing some parasites. However, the amoebolytic capability of these cells was significantly increased in the presence of serum from immune rats. Regardless of the source of serum used, macrophages from immune rats demonstrated about twice the amoebolytic proficiency of cells from naive rats. Macrophages from naive rats showed their highest capacity for lysing amoebae when incubated in the presence of gamma interferon and immune rat serum. The greatest overall proficiency in lysing parasites was displayed by cells from immune rats incubated with A. castellanii in the presence of gamma interferon and nonheat-inactivated serum from immune rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Aerobic salivary bacteria in wild and captive Komodo dragons.

During the months of November 1996, August 1997, and March 1998, saliva and plasma samples were collected for isolation of aerobic bacteria from 26 wild and 13 captive Komodo dragons (Varanus komodoensis). Twenty-eight Gram-negative and 29 Gram-positive species of bacteria were isolated from the saliva of the 39 Komodo dragons. A greater number of wild than captive dragons were positive for both Gram-negative and Gram-positive bacteria. The average number of bacterial species within the saliva of wild dragons was 46% greater than for captive dragons. While Escherichia coli was the most common bacterium isolated from the saliva of wild dragons, this species was not present in captive dragons. The most common bacteria isolated from the saliva of captive dragons were Staphylococcus capitis and Staphylococcus caseolyticus, neither of which were found in wild dragons. High mortality was seen among mice injected with saliva from wild dragons and the only bacterium isolated from the blood of dying mice was Pasteurella multocida. A competitive inhibition enzyme-linked immunosorbent assay revealed the presence of anti-Pasteurella antibody in the plasma of Komodo dragons. Four species of bacteria isolated from dragon saliva showed resistance to one or more of 16 antimicrobics tested. The wide variety of bacteria demonstrated in the saliva of the Komodo dragon in this study, at least one species of which was highly lethal in mice and 54 species of which are known pathogens, support the observation that wounds inflicted by this animal are often associated with sepsis and subsequent bacteremia in prey animals.

The effects of host sex and hormones on Trichinella spiralis in the mouse.

The effects of hosts sex and hormones on the number of adult Trichinella spiralis in the small intestine, the number of migratory larvae produced in vitro by adult female worms, and the number of muscle larvae per gram of body weight were examined in CD-1 Swiss white mice. Nongonadectomized (intact) male mice housed greater numbers of adult worms and a greater number of muscle larvae per gram of body weight than did intact female mice. Adult female worms isolated from intact male mice deposited greater numbers of migratory larvae in vitro than did those obtained from intact female mice. These differences between intact male and intact female mice were eliminated by gonadectomy of male and female mice. Injection of increasing amounts of heterologous sex hormone into intact male and female mice was accompanied by decreasing rates of in vitro larvaposition by adult worms from male mice; but adult worms isolated from female mice showed increasing rates of in vitro larvaposition. Injection of heterologous sex hormone into gonadectomized male and female mice caused a reversal of the findings for intact, uninjected male and female mice stated above. This study has demonstrated that host sex and host sex hormones affect the biology of T. spiralis in the CD-1 Swiss white mouse.

Successful Immunization Against Acanthamoeba Keratitis in a Pig Model

The feasibility of inducing protective immunity to Acanthamoeba keratitis was tested in a pig model. Experiments were designed to determine if ocular infection with Acanthamoeba trophozoites would elicit protection against reinfection. Additional experiments examined whether injection of parasite antigens either intramuscularly, subconjunctivally, or by both routes would induce immunity. Therefore, four groups of animals were examined: (a) pigs that had resolved a primary corneal infection with Acanthamoeba; (b) pigs immunized intramuscularly; (c) pigs immunized subconjunctivally; and (d) pigs immunized intramuscularly and subconjunctivally. Animals were subsequently challenged with parasite-laden soft contact lenses and observed clinically for the appearance of Acanthamoeba keratitis. Acanthamoeba-specific serum antibody titers and blastogenic responses of peripheral blood lymphocytes were determined weekly. The results indicated that intramuscular injection of Acanthamoeba antigens failed to protect against ocular infection even though hosts developed high titers of IgG antibodies and displayed lymphocyte blastogenic responses to parasite antigens. Ocular infection alone failed to stimulate immunity in any of the animals. By contrast, 50% of the hosts immunized subconjunctivally were protected against corneal disease, and 100% of the animals immunized by a combination of intramuscular and subconjunctival administration of parasite antigens were completely protected against two separate ocular challenges with infectious parasites. Protection did not correlate with either IgG antibody titers or blastogenic potentials of peripheral blood lymphocytes. Interestingly, ocular infection alone failed to stimulate immunity to subsequent ocular challenge with infectious parasites. Thus, administration of parasite antigen via the subconjunctival route can protect against Acanthamoeba keratitis.

In vitro and in vivo tumoricidal properties of a pathogenic/free-living amoeba

The chemotactic and tumoricidal properties of the pathogenic/free-living amoeba Acanthamoeba castellanii were examined in vivo and in vitro. A. castellanii trophozoites displayed strong positive chemotactic responses to human melanoma (OCM-1) and murine melanoma (D5.1G4) cells. Although the parasites typically invade and destroy the corneal epithelium and stroma, positive chemotactic responses were not detected against extracts of either of these corneal elements. In vitro studies revealed that viable parasites, as well as cell-free parasite lysates, produced swift and extensive cytolysis of a wide variety of tumor cells. The tumoricidal properties of A. castellanii were examined in vivo and revealed that injection of viable parasites or cell-free parasite lysates into progressively growing subcutaneous melanomas resulted in 83% and 53% reductions in the tumor masses compared with untreated controls. The feasibility of utilizing the tumoricidal properties of pathogenic/free-living amoebae and their cell-free products in the treatment of drug-resistant or radioresistant tumors warrants further investigation.

A role for elevated plasma corticosterone in modulation of host response during infection with Trichinella pseudospiralis

Summary Suppression of host inflammatory response in mice infected with Trichinella pseudospiralis was associated with host plasma corticosterone levels significantly higher than those seen in uninfecled mice or in mice infected with T. spiralis. Increases in the population of mitochondria and depletion of lipid droplets in cells of the zona fasciculata were seen in the adrenals of mice infected with T. pseudospiralis. Elevations in enteritis, myositis and myocarditis accompanied 100% mortality in adrenalectomized mice infected with T. pseudospiralis, while lower levels of inflammation and no mortality were observed in sham operated or intact animals infected with this parasite. The severe myositis normally accompanying infection with T. spiralis was suppressed by concurrent infection with 1000 or 2000 T. pseudospiralis to levels equivalent to those seen in animals receiving 015 and 0‐41 mg cortisone acetate/25 g mouse/day, respectively.

GLUCOSE AND SODIUM FLUXES ACROSS THE BRUSH BORDER OF HYMENOLEPIS DIMINUTA (CESTODA)

When Hymenolepis diminuta was preincubated in Na+-free KRT (tris-maleate buffered Krebs-Ringer saline) for varying time intervals, followed by incubation in 14C-glucose in Na+-fee KRT, the influx of glucose in worms was lowered significantly. This effect of Na+ deletion on glucose influx was totally reversible by incubating worms in KRT ([ Na+] = 154 meq/l).When Na+ in the medium was replaced with K+, tris, or choline, a similar decrease in glucose influx in worms was noted; replacement of Na+ with Li+ resulted in a glucose influx rate significantly higher than that obtained with K+, tris, or choline as the replacement cation. In media with a suboptimal Na+ concentration (25 meq/l), influx of 0.5 mM glucose in worms was unaffected by varying concentrations of K+ (0-100 meq/l).Apparent Michaelis-Menten kinetics were observed when glucose influx as a function of glucose concentration was determined in media with Na+ concentrations of 154, 50, 25, and 10 meq/l. There was a slight decrease in the apparent tra...

Deoxyribonucleic Acid Metabolism in Mouse Trichinosis

Trichinosed mouse diaphragms showed an increase in total RNA, total DNA, and in the incorporation rates of thymidine over that of control muscle. Larval RNA, protein, /Lg RNA per mg protein, and incorporation rates of thymidine were determined. The incorporation of thymidine by infected diaphragms was adjusted for the larval component. Alterations in DNA metabolism are discussed in terms of the morphological changes and changes in RNA metabolism and rates of incorporation of amino acids by infected muscle. Fasske and Themann (1961), and RibasMujal and Rivera-Pomar (1968) have shown that during the early stages of infection with Trichinella spiralis host muscle fibers undergo numerous morphological changes. Read (1970) has reviewed several studies which demonstrate concomitant alterations in the enzymology of infected fibers. These chemical and morphological changes in infected host muscle are accompanied by marked alterations in the total ribonucleic acid (RNA) and rates of synthesis of RNA (Stewart and Read, 1972a). Immediately following a period of rapid RNA turnover there are increases in the rates of incorporation of amino acids by trichinosed muscle (Stewart and Read, 1972b). An increase in the size of nuclei and nucleoli, as well as a proliferation of mitochondria, in infected muscle fibers has been demonstrated by Fasske and Themann (1961) and Ribas-Mujal and Rivera-Pomar (1968). The present study deals with the chronology and magnitude of change in the total deoxyribonucleic acid (DNA) and rates of synthesis of DNA in infected and uninfected mouse

Sarcocystis, Trypanosoma, Toxoplasma, Brugia, Ancylostoma, and Trichinella spp.: A review of the intracellular parasites of striated muscle

Abstract Aspects of the relationship between some protozoan and helminth intracellular parasites of striated muscle and the host cell are reviewed. The first section of the article deals with the myoparasitic protozoa Trypanosoma cruzi, Sarcocystis spp., Toxoplasma gondii, and representatives of the order microsporidia. The second half of the paper considers the myoparasitic helminths Brugia spp., Ancylostoma spp., and Trichinella spiralis. A short description of the life cycle of each parasite is followed by consideration of the mechanisms utilized by the parasite to contact and penetrate the host myofiber. Parasite-induced structural and chemical alterations of the host cell are discussed. Nutrition, growth, and development of the parasite within the host myoenvironment are considered. Finally, conclusions concerning the relationship between the myoparasite and the host muscle fiber are presented.