Susceptibility and features of the ultrastructure of Prototheca zopfii following exposure to copper sulphate,silver nitrate and chlorexidine

One of the most important forms of the occurrence of protothecosis is bovine mastitis. Studies on the in vivo and in vitro susceptibility to antimicrobials have shown that the microorganism is resistant to most of them. Looking for alternative treatments this study aimed to study the susceptibility to copper sulphate (which has an important algicide effect) and silver nitrate (used in dairy cattle breeding for the cauterization of mammary glands) and also to chlorexidine (an important post-dipping anti-septic used in dairy practice), and the effect of these antimicrobials in the ultrastructure of Prototheca zopfii before and after the exposure to these drugs. The in vitro susceptibility tests to chlorexidine, silver nitrate and copper sulphate of the strains of Prototheca zopfii for the determination of their minimal microbicidal concentrations (MMC), were performed using the tube dilution method in Sabouraud dextrose broth and evaluation of colony growth after plating in Sabouraud dextrose agar. The MMCs of chlorexidine, copper sulphate and silver nitrate of the 50 strains tested were 0.01%, 0.1% and 0.3%, respectively. The tubes containing the material used in the antimicrobial susceptibility tests were prepared for the examination in an electron microscope. The untreated controls of P. zopfii showed a similar ultrastructural appearance with the typical characteristics of the microorganism. Cells exposed to silver nitrate showed changes suggesting thickness of the cell wall. Cells exposed to chlorexidine showed changes suggesting degradation of intra-cellular organelles present in the cytoplasm. P. zopfii treated with copper sulphate showed changes suggesting fibrilation of inner layer of cell wall.This revised version was published online in October 2005 with corrections to the Cover Date.     

Evaluation of the susceptibility of Prototheca zopfii to milk pasteurization

Protothecosis has been reported in humans (gastroenteritis, bursitis, etc.) and in many other animal species. Bovine mastitis represents the main form of occurrence of protothecosis in cattle. Milk as well as dairy products, when contaminated with Prototheca spp., represent a potential means of transmission of this zoonosis. The purpose of this study was to evaluate the susceptibility of forty Prototheca zopfii strains isolated from milk from intramammary infections in dairy cows and also from bulk milk tanks of dairy farms, to the different ratios of temperature/time employed in the thermal treatment of milk: 72–75 °C/1 5 seconds, 72–75 °C/20 seconds and 62–65 °C/30 minutes. The samples were subjected to these different temperature/time ratios. The evaluation of the thermal susceptibility of the P. zopfii strains showed that 34 strains were resistant in at least one of the tests. The results point out the need to consider the importance of mastitis caused by Prototheca spp. asrepresenting a public health risk. This revised version was published online in June 2006 with corrections to the Cover Date.     

Susceptibility of multidrug-resistant isolates ofKlebsiella pneumoniae to silver nitrate

Multidrug-resistant (MDR) clinical isolates ofKlebsiella pneumoniae were checked for their sensitivity toward silver nitrate by the tube-dilution method. Nearly 75% of MDR strains clould be successfully inhibited by 5 mg/L of silver nitrate. A significant correlation was observed between incidence of silver and trimoxazole resistance and silver and kanamycin resistance in these isolates. The genetic linkage of these two properties could not be proved since simultaneous curing and co-transfer studies gave negative results.     

Further Observations on the Inhibition of Prototheca zopfii Growth by 3-Amino-l,2,4-triazole

The concentration of amitrole (3-amino-l,2,4-triazole) causing 50 per cent inhibition of Prototheca zopfii growth is 1.25 mg/100 g at 25°C. This inhibition is not reversed by adenine. 50 per cent inhibition of growth is also caused by 100–120 mg/ 100 g 2-aminopurine and this inhibition is partially reversed by 40 mg/100 g adenine, the highest adenine concentration tested due to solubility problems. Imidazoleglycerol accumulation occurs in the medium of cells grown in the presence of amitrole.     

The purification and properties of malonic semialdehyde oxidative decarboxylase from Prototheca zopfii

1. An enzyme, which in the presence of NAD(+) and CoA oxidizes malonic semialdehyde to acetyl-CoA, has been purified from an extract of the colourless alga Prototheca zopfii. 2. The purified enzyme has optimum pH7.5, is specific for NAD(+) and requires a thiol compound for maximum activity. 3. The enzyme is inhibited by arsenite, N-ethylmaleimide and urea. 4. The results are discussed in relation to those obtained by other workers with a similar bacterial enzyme, and a possible reaction sequence is proposed.     

In Vitro Susceptibility of Prototheca to pH and Salt Concentration

Prototheca sp. can assume high economic significance in the dairy industry and pose a potential risk for the public health. We investigated the in vitro susceptibility of Prototheca isolates retrieved from mastitic milk (P. zopfii and P. blaschkeae) to different pH buffers and salt concentrations using a microbroth assay adapted from the Clinical Laboratory Standards Institute guidelines. Different pH buffer solutions ranging from pH 1 to pH 12 and different sodium chloride concentrations, 4.5, 9 and 18%, were tested. P. zopfii strains presented an optimal growth between pH 5 and 9, a complete growth inhibition at pH 3, and limited growth at pH 1 and 12, whereas P. blaschkeae strains showed higher susceptibility to all pH values except for pH 3 where it demonstrated a moderate growth when compared to P. zopfii strains. When salinity was incremented, P. blaschkeae was more resistant than P. zopfii, although a reduction in growth for all strains of Prototheca was observed. This study demonstrated differences in the in vitro susceptibilities of P. zopfii and P. blaschkeae to different pH and salt concentrations and intend to be a contribution on the understanding of some of the physiologic features that can be associated with the survival of these microalgae in the environment.     

The molecular structures of a glucan and a galactan synthesised by Prototheca zopfii

When the unicellular organism Prototheca zopfii was grown on a malt-agar medium, a mixture of polysaccharides was synthesised which could be subsequently extracted from the dried cells with hot water and hot alkali. The major polysaccharide was a galactan which had a branched structure with main chains of (1→6)-linked D-galactopyranose residues, and ≈ 10% of side chains containing terminal D-galacto-furanose residues. A glycogen-type polysaccharide and a (1→4)-linked mannan were also produced.     

Repeated batch cultivation of the hydrocarbon-degrading, micro-algal strain Prototheca zopfii RND16 immobilized in polyurethane foam

This study reports on the stability of the cells of a heterotrophic green micro-algal strain Prototheca zopfii RND16 immobilized in polyurethane foam (PUF) cubes during degradation of mixed hydrocarbon substrate, which was composed of n-alkanes and polycyclic aromatic hydrocarbons (PAHs), in 5 successive cycles of repeated batch cultivation at 30 degrees C. Both RND16 cells and mixed hydrocarbon substrate components had been entrapped in PUF cubes through cultivation. PUF-immobilized RND16 degraded n-alkanes almost completely, whereas the strain hardly degraded PAHs in PUFs, rather they accumulated in the matrices. It is noteworthy that this result is strikingly different from that of the free-living cell culture, where RND16 reduced concentrations of both n-alkanes and PAHs. However, PAHs accumulation in the PUFs did not impair the performance of the immobilized alga to utilize n-alkanes. These results suggest that the PUFs harboring RND16 cells could be used repeatedly for selective retrieval of PAHs from oil-polluted waters after preferential biodegradation of n-alkanes by algae.     

Synthesis of beta-glucans in Prototheca zopfii. Evidence for the existence of a glycoprotein primer

Membrane preparations from the non-photosynthetic alga Prototheca zopfii incorporate glucose from UDP-[3H]glucose into the trichloroacetic-acid-insoluble fraction and the polysaccharides insoluble in hot alkali. Time course and pulse-chase experiments indicate that the acid-insoluble fraction was a precursor of the alkali-insoluble fraction. Isolation of 3H-labeled membrane or soluble fraction showed that only membrane fractions were able to transfer radioactivity into polysaccharides. Treatment of glucosylated membranes with trypsin or cellulase only partially affect their transfer ability, indicating that the precursor was internalized in vesicles. Analysis of the in vitro synthesized polysaccharides by enzymatic and acid hydrolysis showed that glucose and cellobiose were present as radioactive sugars. Permethylation of the polysaccharide indicates that 80% of the glucose was beta-1,4-bonded with 20% in beta-1,3-linkages. This polysaccharide was found to be identical with the cell-wall beta-glucan obtained in vivo [Rivas, L.A. & Pont Lezica, R. (1978) Planta (Berl.) 165, 348-353].     

Histology and ultrastructure of the adult mouse ovary following a single prenatal exposure to diethylstilbestrol

Histology, selective histochemistry and electron microscopy were used to examine the ovarian structure of offspring from mice administered DES (10 micrograms/kg in 0.1 cc of corn oil, subcutaneously) or corn oil alone on Day 15 of gestation. Offspring were sacrificed at 7 months of age. Ovarian changes in DES exposed offspring included the absence of distinguishable corpora lutea but the presence of follicles in various stages of growth and atresia. Large accumulations of pigmented cells and numerous enlarged, pale vacuolated interstitial cells were observed. Interstitial cells contained membrane bound vacuoles, lipid droplets and clumped pigmented material. A concentric pattern of membranes was often observed within the pigment. The results indicate that a single exposure to DES on Day 15 of gestation had a dramatic influence on ovarian morphology and function in 7 month old offspring. The ovarian morphology is consistent with tonic release of FSH and LH and failure in ovulation.     

Effect of Growth Rate on the Macromolecular Composition of Prototheca zopfii, a Colorless Alga Which Divides by Multiple Fission

Prototheca zopfii, a eukaryote that divides by multiple fission, was investigated to determine how growth rate controls daughter cell number. The macromolecular composition, cell size, and number of nuclei per cell were determined in cultures during balanced growth in various media. Cellular mass, ribonucleic acid (RNA), deoxyribonucleic acid (DNA), carbohydrate, and nuclear number increased as positive linear functions of growth rate, whereas nuclear ploidy remained constant with a value of 0.098 pg of DNA/nucleus. The ratios of RNA to protein, protein to mass, and carbohydrate to mass were unaffected by growth rate, whereas the ratios of DNA to protein and RNA to DNA could be expressed as curvilinear functions of growth rate, the former negative and the latter positive. The dependency of normalized gene dosage (DNA/protein) on growth rate appeared as a distinguishing feature of multiple fission. Determination of the normalized rates of protein and RNA synthesis revealed that both increase linearly with growth rate. It is concluded that Prototheca zopfii may exist in a number of physiological states which are characterized by a unique size and macromolecular composition and which are dictated by growth rate.     

The effect of amphotericin B on the ultrastructure of Prototheca species

Prototheca zopfii and Prototheca wickerhamii strains were exposed to subinhibitory concentrations of the antimycotic amphotericin B, and the effect of the treatment on their ultrastructure was assessed. The results revealed ultrastructural changes in the treated cells, expressed by swelling of mitochondria, degradation of cell organelles, accumulation of microbody like structures, lipid droplets and starch granules in the cytoplasm, and changes in the inner layer of the cell wall.     

Sodium turnover rate determines sensitivity to acute copper and silver exposure in freshwater animals

The mechanisms of acute copper and silver toxicity in freshwater organisms appear similar. Both result in inhibition of branchial sodium (and chloride) uptake initiating a cascade of effects leading to mortality. The inhibition of the branchial Na/K-ATPase in the basolateral membrane is generally accepted as the key component responsible for the reduced sodium uptake. We propose that branchial carbonic anhydrase and the apical sodium channel may also be important targets for both copper and silver exposure. Several attempts have been made to predict metal sensitivity. A prominent example is the geochemical-biotic ligand model. The geochemical-biotic ligand modeling approach has been successful in explaining variations in tolerance to metal exposure for specific groups of animals exposed at different water chemistries. This approach, however, cannot explain the large observed variation in tolerance to these metals amongst different groups of freshwater animals (i.e. Daphnia vs. fish). Based on the detailed knowledge of physiological responses to acute metal exposure, the present review offers an explanation for the observed variation in tolerance. Smaller animals are more sensitive than large animals because they exhibit higher sodium turnover rates. The same relative inhibition of sodium uptake results in faster depletion of internal sodium in animals with higher sodium turnover. We present a way to improve predictions of acute metal sensitivity, noting that sodium turnover rate is the key predictor for variation in acute copper and silver toxicity amongst groups of freshwater animals. We suggest that the presented sodium turnover model is used in conjunction with the Biotic Ligand Model for risk management decisions.     

Expression of bovine lactoferrin and lactoferrin N-lobe by recombinant baculovirus and its antimicrobial activity against Prototheca zopfii.

Lactoferrin (LF) is a multifunctional, iron-binding glycoprotein found in secretory fluids of mammals. In this study, DNA encoding bovine lactoferrin (bLF) or the N-terminal half of bLF (bLF N-lobe) was inserted into a baculovirus transfer vector, and a recombinant virus expressing bLF or bLF N-lobe was isolated. An 80-kDa bLF-related protein expressed by the recombinant baculovirus was detected by monoclonal antibodies against bLF N-lobe and the C-terminal half of bLF (bLF C-lobe). A 43-kDa bLF N-lobe-related protein expressed by the recombinant baculovirus was detected by anti-bLF N-lobe monoclonal antibody, but not by anti-bLF C-lobe monoclonal antibody. These proteins were also secreted into the supernatant of insect cell cultures. Recombinant bLF (rbLF) and bLF N-lobe (rbLF N-lobe) were affected by tunicamycin treatment, indicating that rbLF and rbLF N-lobe contain an N-linked glycosylation site. Antimicrobial activity of these recombinant proteins against Prototheca zopfii (a yeast-like fungus that causes bovine mastitis) was evaluated by measuring the optical density of the culture microplate. Prototheca zopfii was sensitive to rbLF and rbLF N-lobe, as well as native bLF. There was no difference in antimicrobial activity between rbLF N-lobe and bLF C-lobe.