Viability of Giardia cysts suspended in lake, river, and tap water

Numerous waterborne outbreaks of giardiasis have occurred since 1965, yet little or no information has been reported on the viability of Giardia cysts in different aquatic environments. We have studied the viability of Giardia muris cysts suspended in lake, river, and tap water, while also monitoring water temperature, dissolved oxygen, pH, and other water quality parameters. Fecal pellets containing G. muris cysts were placed in glass vials covered with filter paper and exposed to (i) lake water at 15 ft (ca. 4.6 m) and 30 ft (ca. 9.2 m), (ii) river water, (iii) tap water, and (iv) distilled water stored under laboratory conditions. At 3, 7, 14, 28, 56, and 84 days, two vials from each environment were removed, and cyst viability was determined by (i) fluorogenic dye exclusion, (ii) production of giardiasis in an animal, and (iii) cyst morphology by Nomarski microscopy. In the fall, the cysts suspended at 30 ft in lake water remained viable for up to 56 days whereas cysts stored at 15 ft were nonviable after day 28. The G. muris cysts exposed to river water remained viable up to 28 days as determined by the production of giardiasis in mice. G. muris cysts suspended in tap water showed no signs of viability after 14 days, while cysts serving as controls (exposed to refrigerated distilled water) remained viable for up to 56 days. In the winter, Giardia cysts suspended in either lake or river water were viable for 56 to 84 days whereas cysts exposed to tap water were nonviable by day 14.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cross-species transmission of Giardia spp.: inoculation of beavers and muskrats with cysts of human, beaver, mouse, and muskrat origin

Giardia cysts isolated from humans, beavers, mice, and muskrats were tested in cross-species transmission experiments for their ability to infect either beavers or muskrats. Giardia cysts, derived from multiple symptomatic human donors and used for inoculation of beavers or muskrats, were shown to be viable by incorporation of fluorogenic dyes, excystation, and their ability to produce infections in the Mongolian gerbil model. Inoculation of beavers with 5 x 10(5) Giardia lamblia cysts resulted in the infection of 75% of the animals (n = 8), as judged by the presence of fecal cysts or intestinal trophozoites at necropsy. The mean prepatent period was 13.1 days. An infective dose experiment, using 5 x 10(1) to 5 x 10(5) viable G. lamblia cysts collected by fluorescence-activated cell sorting, demonstrated that doses of between, less than 50, and less than 500 viable cysts were required to produce infection in beavers. Scanning electron microscopy of beaver small intestine revealed that attachment of G. lamblia trophozoites produced lesions in the microvillous border. Inoculation of muskrats with G. lamblia cysts produced infections when the dose of cysts was equal to or greater than 1.25 x 10(5). The inoculation of beavers with Giardia ondatrae or Giardia muris cysts did not produce any infection; however, the administration to muskrats of Giardia cysts of beaver origin resulted in the infection of 62% of the animals (n = 8), with a prepatent period of 5 days. Our results demonstrated that beavers and muskrats could be infected with Giardia cysts derived from humans, but only by using large numbers of cysts.(ABSTRACT TRUNCATED AT 250 WORDS)     

High-resolution immunogold localization of Giardia cyst wall antigens using field emission SEM with secondary and backscatter electron imaging

We describe here the ultrastructural localization of Giardia cyst antigens in the filaments associated with the outer portion of intact cysts and on developing cyst wall filaments in encysting trophozoites. Post-embedding immunogold labeling of thin sections of intact Giardia cysts with polyclonal and monoclonal antibodies specific for cyst wall antigens (major protein bands of approximately 29, 75, 88, and 102 KD on Western blots) showed strong labeling of the filamentous cyst wall, whereas no labeling was seen on the membranous portion. High-resolution field emission scanning electron microscopy (FESEM) of Giardia cysts revealed that the cyst wall-specific polyclonal rabbit antisera and monoclonal mouse antibody produced gold labeling of 20-nm filaments in the cyst wall as detected with secondary electron imaging (SEI) and backscatter electron imaging (BEI) at 10 kV, despite coating of the cells with platinum by ion sputtering. FESEM studies of encysting Giardia trophozoites demonstrated that immunostaining with antibodies to cyst wall antigens produced colloidal gold labeling of developing cyst wall filaments on the cell surface; however, the intervening membrane domains were unlabeled. Substitution of normal serum for cyst wall-specific antibodies, or preabsorption of specific antibodies with Giardia cysts, eliminated immunolabeling of the filaments.     

Hypomethylated sequences: Characterization of the duplicate soybean genome

Soybean is believed to be a diploidized tetraploid generated from an allotetraploid ancestor. In this study, we used hypomethylated genomic DNA as a source of probes to investigate the genomic structure and methylation patterns of duplicated sequences. Forty-five genomic clones from Phaseolus vulgaris and 664 genomic clones from Glycine max were used to examine the duplicated regions in the soybean genome. Southern analysis of genomic DNA using probes from both sources revealed that greater than 15% of the hypomethylated genomic regions were only present once in the soybean genome. The remaining ca. 85% of the hypomethylated regions comprise duplicated or middle repetitive DNA sequences. If only the ratio of single to duplicate probe patterns is considered, it appears that 25% of the single-copy sequences have been lost. By using a subset of probes that only detected duplicated sequences, we examined the methylation status of the homeologous genomes with the restriction enzymes MspI and HpaII. We found that in all cases both copies of these regions were hypomethylated, although there were examples of low-level methylation. It appears that duplicate sequences are being eliminated in the diploidization process. Our data reveal no evidence that duplicated sequences are being silenced by inactivation correlated with methylation patterns.     

Slow cortical potential biofeedback and the startle reflex

The negativity of slow cortical potentials (SCP) of the surface EEG is a measure of brain excitability, correlating with motor and cognitive preparation. Selfcontrol of SCP positivity has been shown to reduce seizure activity. Following SCP biofeedback from a central EEG electrode position, subjects gained bidirectional control over their SCP. The current study used a modified feedback methodology, and found a positive relationship between negativity and magnitude of EMG startle response (a measure of cortical and subcortical arousal, particularly aversive response disposition). Greater success in SCP differentiation was associated with self-report of less relaxation during negativity training.This research was supported by the Deutsche Forschungsgemeinschaft under grant No. SFB 307.     

An Ustilago maydis Mutant Partially Blocked in P45014DM Activity Is Hypersensitive to Azole Fungicides

An Ustilago maydis ergosterol biosynthesis mutant (A14) which is partially blocked in sterol 14alpha-demethylase (P45014DM) activity is described. This mutant accumulated the abnormal 14alpha-methyl sterols, eburicol, 14alpha-methylfecosterol, and obtusifoliol, along with significant amounts of ergosterol. Although the A14 mutant grew nearly as well as the wild type, it was impaired in cell extension growth, which indicated a dysfunction in apical cell wall synthesis. The mutant was also found to be hypersensitive to the azole fungicides penconazole and tebuconazole.     

Esterase-16 (Es-16): Characterization,polymorphism, and linkage to chromosome 3 of a kidney esterase locus of the house mouse

A polymorphism for an isozyme of a presumed arylesterase, esterase-16 (EC 3.1.1.2), has been detected in kidney, heart, and spleen of the house mouse, Mus musculus, by means of isoelectric focusing and by disc electrophoresis. Three phenotypes can be distinguished: the ES-16A phenotype (IEP 5.9) was found in C57BL/10Sn and many other laboratory inbred strains; the ES-16B phenotype (IEP 6.1) was found in M. m. molossinus; and the ES-16C phenotype (IEP 5.9; very weak activity) was found in Peru-Coppock. Esterase-16 is strongly inhibited by 10^?3 m p-chloromercuribenzoate, but not by 2·10^?4 m bis-p-nitrophenyl phosphate or by 10^?3 m Diamox. It stains well with indoxyl acetate and other indigogenic substrates but only weakly with α-naphthyl acetate. Esterase-16 is completely insoluble in water. It is apparently governed by a structural gene locus, Es-16, with three alleles, Es-16 ^a , Es-16^b, and Es-16 ^c, respectively. Es-16 is closely linked to Car-1 and Car-2 on chromosome 3. Typing of 94 animals of the backcross (C57BL/10Sn × M. m. mol.) F₁ × M. m. mol. revealed a recombination frequency of 8.51±2.9%.     

Hexazonium pararosaniline as a fixative for animal tissues

Hexazonium pararosaniline is a valuable reagent that has been used in enzyme activity histochemistry for 50 years. It is an aqueous solution containing the tris-diazonium ion derived from pararosaniline, an aminotriarylmethane dye, and it contains an excess of nitrous acid that was not consumed in the diazotization reaction. Other investigators have found that immersion for 2 min in an acidic (pH 3.5) 0.0015 M hexazonium pararosaniline solution can protect cryostat sections of unfixed animal tissues from the deleterious effects of aqueous reagents such as buffered solutions used in immunohistochemistry, while preserving specific affinities for antibodies. In the present investigation hexazonium pararosaniline protected lymphoid tissue and striated muscle against the damaging effects of water or saline. The same protection was conferred on unfixed sections treated with dilute nitrous or hydrochloric acid in concentrations similar to those in hexazonium pararosaniline solutions. Model tissues (solutions, gels or films containing gelatin and/or bovine albumin) responded predictably to well known cross-linking (formaldehyde) or coagulant (mercuric chloride) fixatives. Hexazonium pararosaniline solutions prevented the dissolution of protein gels in water only after 9 or more days of contact, during which time considerable swelling occurred. It is concluded that there is no evidence for a “fixative” action of hexazonium pararosaniline. The protective effect on frozen sections of unfixed tissue is attributable probably to the low pH of the solution.