Toxicity of Diplodia maydis to laboratory animals.

Ten strains of Diplodia maydis isolated from commercial corn samples and grown on whole yellow corn at 25 degrees C for 6 weeks were toxic to ducklings and rats. The degree of toxicity depended on the incubation period and temperature. Minimum incubation periods of 3 to 4 weeks and 6 weeks were necessary to cause mortality in ducklings and rats, respectively. Cultures incubated at 31 and 25 degrees C were much more toxic than those kept at 16 and 20 degrees C. Heat treatments at 80 to 90 degrees C destroyed most of the toxicity of moldy meal. Mild lesions of a similar histopathological nature were found in subclinically and lethally poisoned rats. These included toxic myocarditis, enteritis, focal renal tubular necrosis, degeneration and peripheral necrosis of the islets of Langerhans, and a generalized venous congestion.     

Ultrastructure and elemental composition of dormant and germinating Diplodia maydis spores.

The ultrastructure of Diplodia maydis spores was studied in thin sections with a transmission electron microscope. Storage vacuoles were evenly distributed in the two cells. Some of the vacuoles that contained a dense osmiophilic sphere(s) were surrounded by a membrane, and had membranous aggregates around their periphery. The sport wall was composed of an electron-dense layer and an electron-translucent layer. An inner cytoplasmic membrane was present. Dormant and germinating spores were studied with scanning electron microscopy and also with a Si (Li) energy-dispersive X-ray analyzer. The dormant spore was ovate and usually two-celled with a central septum. Germination proceeded via a germ tube from the side of one end of the cell. Of several methods for preparation of specimens for X-ray analysis studied, freeze-dried spores mounted on carbon stubs and then further carbon coated gave the best results. X-ray analyses revealed that spore populations contained large amounts of Si, P, Cl, and K, smaller amounts of S and Ca, and trace amounts of Mg and Al. Analyses of single spores revealed high K and Cl and low P and Mg at one end of the cell with concomitant low K and Cl and high P and Mg in the central portion and other end of the cell. In two-celled germinating spores, high K and Cl occurred in the end of the nongerminating spore cell, whereas the germinating cell contained high P and Mg and low K and Cl. X-ray image maps revealed that K and Cl were located together at one end of the spore.     

Dry mass changes in germinating spores of Diplodia maydis

Summary The dry mass of two-celled Diplodia maydis spores was measured both before and after germination by quantitative interference microscopy. The dry mass of spores declined approximately 50% during germination. However, the dry mass of germinating spores plus the dry mass of their germ tubes was greater than the dry mass of spores before germination. We conclude that the germinating spores absorbed nutrients released from non-germinating spores.The dry mass of fungal spores can be estimated by weighing large numbers of spores and determining the mean from sample spore counts. Mumford and Pappelis(4) determined the total dry mass of individual spores of Fusarium roseum and the contained lipid bodies before and after spores germinated using quantitative interference microscopy. The mean spore dry mass before germination was 57 pg. Lipid bodies accounted for about 61% of that mass and decreased as spores germinated. The total dry mass of the spore and germ tube 24 hr later greatly exceeded that of the spore before germination. Quantitative interference microscopy has been used to measure the dry mass of various types of cells. Kulfinski and Pappelis (3) recently reviewed how this technique has been applied to plant cells. Technical aspects of interference microscopy have been described by Ross (6).The purpose of this study was to examine the dry mass changes in Diplodia maydis (Berk.) Sacc. with and without germ tubes through the use of interference microscopy.     

Ultrastructural Histopathology of Infection and Colonization of Maize by Stenocarpella maydis (= Diplodia maydis)

The mode of penetration and colonization of stalk, shank and leaf sheath tissues of maize by Stenocarpella maydis (=Diplodia maydis) was determined by Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM) observations. Detached plant tissues, inoculated with a conidial suspension and examined by SEM at various intervals, showed that S. maydis conidia germinated on all plant material types after 5 h incubation at 30 °C. After 72 h incubation, appressoria had formed at the hyphal tips. Similar observations were recorded on plants inoculated in the glasshouse except that germination was delayed by 7 days after inoculation. TEM studies showed that penetration was affected by a penetration hypha which resulted in the inter- and intracellular colonization of the plant tissues. Colonization was accompanied by degradation of cell walls suggesting that host colonization is facilitated by enzyme activity.     

Ultrastructure of Diplodia maydis grown on selected synthetic media

Of all the fixatives studied, glutaraldehyde followed by osmium was considered best. Fixation temperatures did not result in ultrastructural differences. Holdover for short periods (up to 7 days) in 70% alcohol and long periods (3 months) in glutaraldehyde did not result in ultrastructural differences from those processed without a hold-over period. Uranyl acetate increased contrast, as expected. Epon was selected over Epon-Araldite (more viscous) and Spurr's resin (less contrast). Generally, growth conditions did not alter gross hyphal morphology, although cytological changes were noted. Diplodia maydis is morphologically similar to ascomycetous fungi. It contained typical organelles as found in other fungi, e.g. cell wall, septa, Woronin bodies, nuclei, mitochondria, endoplasmic reticulum, lomasomes, plasma membranes, cytosomes, vacuoles, storage materials (lipids, carbohydrates and intracellular and extracellular electrondense substances), thickened mycelial strands, and intrahyphal hyphae. Bead-like structures and thickened nodes as observed by SEM were accounted for by structures observed by TEM. Mitochondria were dramatically elongated, and in one case, branched. Hyphae were organized in three zones: apical, subapical, and vacuolated. Apical vesicles were thought to account for cell extension. Evidence was presented that Woronin bodies originated from microbodies. It was suggested that the Golgi apparatus consists of isolated single cisternae. Free ribo- or polysomes (without ER) were suggested as the site of the majority of protein synthesis. Continuities and configurations of plasma and outer nuclear and mitochondrial membranes, ER, cytosomes, vacuoles and storage materials suggested that a system similar to Morre et al's endomembrane system is in operation in D. maydis to account for component flow and differentiation.     

Nitrogen-metabolizing enzymes of Diplodia maydis, a Zea mays L. stalk rot causing fungus.

The nitrogen source available to Diplodia maydis in vivo is reported to affect the severity of stalk rot in maize. Nitrate and (or) ammonium salts were tested for their effect on the type of nitrogen metabolism found in Diplodia maydis in vitro. The level of glutamate dehydrogenase remained essentially constant on either nitrogen salt but nitrate reductase was induced by growth on nitrate salts and was not extractable on ammonium salts. Properties of nitrate reductase reported here are similar to those reported for the higher plant and Neurospora crassa enzymes. Thr relationship of nitrogen metabolism in Diplodia maydis to Zea mays L. stalk rot is discussed.     

Toxicity of tobramycin in single and multiple administrations to laboratory animals]

The LD50 of tobramycin sulphate administered intravenously, intraperitoneally, subcutaneously and orally to albino mice was 77 (73--82), 262 (234--294), 560 (500--627) and greater than 10500 mg/kg respectively. With an increase in the rate of intravenous administration tobramycin toxicity increased. When tobramycin sulphate was administered subcutaneously daily in multiple doses equivalent to the daily therapeutic doses from humans (calculated for the body surface) and in the doses 2--3 times higher than the above therapeutic ones, the function of the kidneys, liver and the Preier's reflex did not significantly change. When the doses were 8--10 times higher than the therapeutic ones, an increase in the urea level in the blood serum, disappearance and a decrease in the Preier's reflex were observed. The impairment of the kidney function was accompanied by degenerative changes in the convoluted tubules of the kidneys, ischemia of the renal glomeruli and appearance of protein secretion in their capsule cavities. The picture of the peripheral blood did not suffer significant changes. The studies on the acute and chronic toxicity of tobramycin sulphate prepared at the Institute of New Antibiotics showed that the drug did not differ from the import tobramycin samples.     

Mycotoxic peripheral myelinopathy, myopathy, and hepatitis caused by Diplodia maydis in vervet monkeys.

During recent historical times many Africans changed their diet to one based on maize. The grain is regularly contaminated by fungi which are toxigenic to domestic animals and birds. After one of the fungi, Diplodia maydis, in pure culture on maize, was added to the food of omnivorous primates there was demyelination of nerves, atrophy, degeneration and necrosis of muscle, and hepatitis. These preliminary results are applicable to veterinary and laboratory animal sciences. They may also be medically significant since neuromuscular syndromes of unknown cause are prevalent among Africans. Nerve conduction velocities and organ weights are defined for vervet monkeys.     

Studies of the Toxicity of Helminthosporium maydis

Isolates of Helminthosporium maydis from blighted corn were tested for toxicity in mice, rats, swine, rabbits, microorganisms, and tissue culture. Extracts of grains, mycelia, and culture supernatant fluids killed mice on intraperitoneal (ip) injection, but were nontoxic on administration by mouth to swine. The toxin was partially purified and appears to be a glycophospholipid. Histopathological examination revealed that the toxin acted as a severe irritant on ip injection, causing death in laboratory animals. In skin tests with rabbits, considerable exudation occurred, rather than necrosis.     

The fungus Ustilago maydis, from the aztec cuisine to the research laboratory.

Ustilago maydis is a plant pathogen fungus responsible for corn smut. It has a complex life cycle. In its saprophitic stage, it grows as haploid yeast cells, while in the invasive stage it grows as a mycelium formed by diploid cells. Thus, a correlation exists between genetic ploidy, pathogenicity and morphogenesis. Dimorphism can be modulated in vitro by changing environmental parameters such as pH. Studies with auxotrophic mutants have shown that polyamines play a central role in regulating dimorphism. Molecular biology approaches are being employed for the analysis of fundamental aspects of the biology of this fungus, such as mating type regulation, dimorphism or cell wall biogenesis.     

Morphological Observations of Diplodia maydis on Synthetic and Natural Substrates as Revealed by Scanning Electron Microscopy

Mycelial and spore morphology of Diplodia maydis were investigated by using scanning electron microscopy after growth on various media and natural substrates (oat and corn kernels, and corn husks). Of several specimen preparation methods studied, Parducz fixation followed by critical-point or freeze-drying gave adequate preservation for pycnidia, mycelia, and spores. Morphological characteristics were similar in rotary and reciprocal shaker cultures and differed from that found in stationary cultures in the amount of slime-like material produced and precipitated matter on the mycelial surfaces. In general, mycelial surfaces were smooth. Large areas of coalesced material were present in all samples examined. Slime-like material produced in liquid media appeared as a finely laced net, randomly appearing throughout the mycelia with bead-like structures present along the net. A fine netting also was observed interspersed among the spores inside the pycnidia obtained from oats. Slime-like material was observed to cover the pycnidia produced on oat and corn kernels. In the latter case, the spores were less protected by the outer slime-like covering. Thickened node-like structures were observed in mycelial mats produced in modified Fries 2 medium, on potato dextrose agar plates, and on infected oats. Round and ovate thickened node-like structures were observed in mycelium produced on corn kernels. In general, node-like structures were less abundant in mycelia from naturally infected substrates. Conidia were commonly rounded to tapered and two celled, with a distinctive ridged septum at the middle. Dried spores were collapsed in a characteristic flask-like fashion.     

Pharmacokinetics of fusidic acid in laboratory animals.

The ability of evaluate the efficacy of fusidic acid in animal models of infectious disease is limited by the absence of pharmacokinetic data for the agent in laboratory animals. In our study, aspects of fusidic acid pharmacokinetics were compared in rats (Rattus norwegicus), mice (Mus musculus), rabbits (Oryctolagus cuniculus), and guinea pigs (Cavia porcellus). Sodium fusidate was poorly absorbed after oral administration to rats, although limited absorption occurred in guinea pigs, mice, and rabbits. Subcutaneous injections of diethanolamine fusidate to laboratory rats, however, achieved a serum profile similar to that observed in humans. There was no evidence of drug accumulation in rats given repeated subcutaneous doses of diethanolamine fusidate during a 4-day period, but rabbits showed clear evidence of a cumulative effect.     

Wallace P. Rowe lecture. Poxviruses of laboratory animals

Currently the subfamily Chordopoxvirinae, the poxviruses of vertebrates, is subdivided into eight genera, containing some 20-30 species; an inexact figure because the birdpox viruses have not yet been properly investigated taxonomically. I have discussed seven species belonging to three genera, all of which have caused infection and usually disease in mammals commonly used in the laboratory. The list could have been extended had I included chickens and swine as laboratory animals, for that would have meant that I would have spoken about the birdpox viruses and swinepox virus as well. However, I think I have said enough to remind you of the importance of this family of viruses to those of you concerned with laboratory animal medicine. I believe that Wally Rowe would have been interested, for every case I have described presents problems in the ecology of viruses, and like my mentor Macfarlane Burnet, Wally approached virology from an ecological point of view, whether he was thinking about the DNA provirus of retroviruses and the host chromosome, the pathogenesis of disease, or the spread of viruses in animal populations, all topics to which he made major contributions.     

An anesthetic system for small laboratory animals.

A non-rebreathing anesthetic system for administering volatile anesthetic mixtures to small laboratory animals was designed. The device is easily constructed and simple to operate, yet permits the anesthetic management of small animals by technics similar to those used for man and larger species.     

An apparatus for anesthetizing small laboratory animals.

A simple apparatus was designed for simultaneous general anesthesia of a number of small laboratory animals with spontaneous respiration. Anesthesia is consistent over many hr. The apparatus consists of an oxygen tank and vaporizer, a glass distribution bottle, and a non-return circuit of separate inspiratory and expiratory valves for each animal. The equipment is inexpensive to construct and can be rapidly adjusted for exact control of gas flow and anesthetic depth.