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The type species of Prototheca stagnora, SEC-L-1690 (ATCC 16528) (2) was found to have a large capsule surrounding the cells, in India ink preparations. Cryptococcus neoformans and P. stagnora are very similar organisms in their morphology and they should be differentiated by their reproduction process or by their biochemical and physiological characteristics.
In the last 2 years 223 cases of bovine mastitis caused by Prototheca zopfii infection were identified in 32 large-scale dairy herds. All of these farms were in Hungary, which has a continental type, temperate zone climate. Both the sporadic and epidemic forms of P. zopfii mastitis were observed. All the herds affected by the epidemic form had poor hygienic conditions and suffered from several managerial faults, but no specific predisposing factors could be identified. In almost all of the cases, the type II variant of this pathogen was isolated; however, the type III variant was isolated from three cows. The cows had a higher chance of new infection in the early weeks of lactation and in the summer. The P. zopfii infection usually resulted in a chronic subclinical, or mild clinical, inflammatory process in the udder, and was followed by a dramatic loss in milk production and a permanent increase in somatic cell count. The histopathological findings could be characterized as a progressive interstitial mastitis associated with alveolar atrophy. The self-recovery rate was very low.
Prototheca zopfii, an alga-like organism, was isolated from subungual material in a patient with onycholysis. This organism seldom causes human infections. Clinical and laboratory aspects are described. The literature is reviewed.
A 72-year-old man who was undergoing chronic ambulatory peritoneal dialysis developed peritonitis due to Prototheca wickerhamii. Results of in vitro sensitivity testing revealed that the organism was sensitive to amphotericin B (MIC, 0.4 micrograms/mL) but resistant to fluconazole (MIC, greater than 80 micrograms/mL). The patient was successfully treated medically with a combination of intravenous amphotericin B and oral doxycycline.
Protothecosis is an uncommon infection due to Prototheca wickerhamii. Recently, a patient with progressive cutaneous protothecosis and an apparent defect in the ability of her polymorphonuclear neutrophils (PMNs) to kill P. wickerhamii was successfully treated. Therefore, the role of PMNs and serum opsonins in host defense against tissue invasion by this organism was evaluated. The results show that human PMNs ingested and kill P. wickerhamii. Ultrastructural studies revealed digestion of the organism by PMNs 60 min after ingestion. Optimal phagocytosis and killing of P. wickerhamii by PMNs required the presence of both specific IgG antibody and heat-labile opsonins.
When mutant strain UV77-247 of Prototheca moriformis Kruger was fed d-[1-13C]Glc, it synthesized l-ascorbic acid (AA) with approximately three-quarters of the label at the C-1 position and the remaining label at the C-6 position, showing that AA is made by a non-inversion (retention) pathway, i.e. C-1 of Glc becomes C-1 of AA. The label present at C-6 is consistent with the glycolytic conversion of Glc to 3-carbon intermediates and subsequent gluconeogenesis. Compounds suggested as intermediates in inversion-type pathways were not converted to AA. Most strains converted Man to AA at a rate greater than they did Glc. Enzyme activities leading from Fru-6-P to the formation of GDP-Man were identified in all strains, but none of these activities correlated with the mutants' abilities to accumulate AA. However, there was a strong correlation between GDP-Man-3,5-epimerase activity and AA accumulation. Wild-type P. moriformis ATCC 75669 and mutant strains of varying AA-synthesizing abilities rapidly converted l-Gal or l-galactono-1,4-lactone to AA. Based on this data, a biosynthetic pathway from Glc to AA is proposed in which the epimerase is the rate-limiting activity in AA synthesis.
The mitochondrial DNA from the colourless alga Prototheca wickerhamii contains two mosaic genes as was revealed from complete sequencing of the circular extranuclear genome. The genes for the large subunit of the ribosomal RNA (LSUrRNA) as well as for subunit I of the cytochrome oxidase (coxI) carry two and three intronic sequences respectively. On the basis of their canonical nucleotide sequences they can be classified as group I introns. Phylogenetic comparisons of the coxI protein sequences allow us to conclude that the P.wickerhamii mtDNA is much closer related to higher plant mtDNAs than to those of the chlorophyte alga C.reinhardtii. The comparison of the intron sequences revealed several unusual features: (1) The P.wickerhamii introns are structurally related to mitochondrial introns from various ascomycetous fungi. (2) Phylogenetic analyses indicate a close relationship between fungal and algal intronic sequences. (3) The P. wickerhamii introns are located at positions within the structural genes which can be considered as preferred intron insertion sites in homologous mitochondrial genes from fungi or liverwort. In all cases, the sequences adjacent to the insertion sites are very well conserved over large evolutionary distances. Our finding of highly similar introns in fungi and algae is consistent with the idea that introns have already been present in the bacterial ancestors of present day mitochondria and evolved concomitantly with the organelles.
A representative selection of Prototheca zopfii strains isolated from different environmental habitats or clinical cases was characterized in a polyphasic approach in order to assess their intraspecies taxonomic position. Recently, the recognition of distinct phenotypic clusters has been reported as the assignment of 'variants'. In the present study, 11 strains were compared by a number of phenotypic and genetic criteria, including growth characteristics, biochemical reactions and serotyping results. Based on emended standards for biotype identification, P. zopfii strains showed auxanographic differences and distinct assimilation patterns with respect to utilization of amino acids and glycerol. Serotyping by means of immunoblotting revealed that all isolates of variant II obtained from clinical cases, i.e. isolates from bovine mastitis or from human enteropathia, showed specific antigen patterns. They were found to be different from strains assigned to the other two variants with respect to their immunogenic antigens. Furthermore, comparison of partial 18S rDNA sequences confirmed distinct differences between the former variants. Based on these results, it is proposed that P. zopfii merits classification as a species comprising three biotypes.
Electron microscopic analysis was conducted on epithelioid cell granulomas experimentally induced by prototheca in the skin of mice. In BALB/c mice, epithelioid cell granulomas were easily induced. Each cell had a round nucleus with euchromatin on the margin, abundant cytoplasmic organelles, and particularly abundant rough endoplasmic reticulum (rER). Cells with a plasmacytoid appearance were the most common. Fibroblast-like epithelioid cells were sometimes observed. The whole body and cell membrane of an organism was usually within the cytoplasm of epithelioid cell. In ICR mice, epithelioid cell granulomas were not easily induced. Cells which had ingested whole organisms had spindle shaped and/or oval nuclei with marginated heterochromatin and with abundant dilated rER. Near completion of organism digestion, the cells had irregular and/or indented nuclei with marginated heterochromatin and often one or two distinct nucleoli. The cytoplasm was often replaced with numerous phagocytic vacuoles. At higher magnification, secondary lysosomes and Golgi complexes forming Golgi lamellae could be seen in the cells in which cytoplasmic organelles such as endoplasmic organelles and mitochondria were abundantly present. In contrast to the BALB/c mice, the remnants of digested organisms, split up into many small pieces, could be seen in the cytoplasm. The cells with a fibroblast-like appearance were most frequent, but plasmacytoid cells were sometimes observed. In the present study, the ultrastructural features of epithelioid cells in BALB/c and ICR mice clearly differed. Plasmacytoid cells were the most abundant in BALB/c mice, while fibroblast-like cells were in ICR mice.
The lipid composition of Prototheca wickerhamii ATCC 16529 is presented and discussed in relation to the unique susceptibility of the organism to drugs of three membrane-active antimicrobial classes: the polyenes, the polymyxins, and the imidazoles. The presence of ergosterol in the neutral lipid fraction of the membrane is likely responsible for the exquisite susceptibility to amphotericin B. The presence of a large quantity of free fatty acids in the membrane appears responsible for imidazole susceptibility. The membrane determinants of polymyxin B susceptibility are less well defined.
The presence of plastids in diverse eukaryotic lineages that have lost the capacity for photosynthesis is well documented. The metabolic functions of such organelles, however, are poorly understood except in the case of the apicoplast in the Apicomplexa, a group of intracellular parasites including Plasmodium falciparum, and the plastid of the green alga Helicosporidium sp., a parasite for which the only host-free stage identified in nature so far is represented by cysts. As a first step in the reconstruction of plastid functions in a nonphotosynthetic, predominantly free-living organism, we searched for expressed sequence tags (ESTs) that correspond to nucleus-encoded plastid-targeted polypeptides in the green alga Prototheca wickerhamii. From 3,856 ESTs, we found that 71 unique sequences (235 ESTs) correspond to different nucleus-encoded putatively plastid-targeted polypeptides. The identified proteins predict that carbohydrate, amino acid, lipid, tetrapyrrole, and isoprenoid metabolism as well as de novo purine biosynthesis and oxidoreductive processes take place in the plastid of P. wickerhamii. Mg-protoporphyrin accumulation and, therefore, plastid-to-nucleus signaling might also occur in this nonphotosynthetic organism, as we identified a transcript which encodes subunit I of Mg-chelatase, the enzyme which catalyzes the first committed step in chlorophyll synthesis. Our data indicate a far more complex metabolism in P. wickerhamii's plastid compared with the metabolic pathways predicted to be located in the apicoplast of P. falciparum and the plastid of Helicosporidium sp.
Prototheca wickerhamii is a rare cause of systemic infection in humans. While some cases occur in previously healthy individuals, others are associated with a variety of preexisting diseases. Here we present, for the first time, a case of P. wickerhamii algaemia in a patient with myasthenia gravis. The patient was successfully treated with amphotericin B.
In order to characterize the humoral and cellular immune responses to bovine mammary protothecosis, serum and whey samples obtained from 72 dairy cows assigned to four different clinical stages of infection were examined for specific antibodies by indirect enzyme-linked immunosorbent assay techniques. Milk samples were analyzed for the total numbers of excreted algal cells and somatic cells. After characterization of the course of immune induction in bovine protothecal mastitis, a long-term sentinel study was performed in an affected herd in order to investigate disease progression. A total of 61 dairy cows with protothecal mastitis were examined for shedding of algae cells and for local immune responses three times in 6-month intervals. During acute and chronic stages of protothecosis, significantly elevated specific antibody activities in sera were detected. A strong correlation of whey immunoglobulin A (IgA) and whey IgG1 antibody activity with the total counts of somatic cells in milk was observed, whereas only a weak correlation of whey IgA and whey IgG1 concentrations to the number of algal cells excreted with the milk was seen. Our results from the sentinel long-term study of infected cows revealed that 70.5% of the persistently infected animals were continuously shedding the pathogen. About 4.9% of the animals showed an intermittent shedding, whereas 18% of the cows were tested culturally negative throughout the study. It can be assumed that Prototheca zopfii mastitis in dairy cows is maintained on the herd level by subclinically infected alga-shedding cows.
Disseminated infection with Prototheca zopfii is a rare disease in immunosuppressed patients. We here report the first case of lethal infection with P. zopfii following unrelated stem cell transplantation for leukemia. Breakthrough protothecosis occurred during long-term administration of voriconazole in the case of pulmonary aspergillosis.
An outbreak of severe indurative mastitis in newly calved cows, from which Prototheca zopfii was isolated, is described. The refractory nature of this type of mastitis and the ineffectiveness of the presently available intramammary preparations are highlighted.
Mammary glands from cows with protothecal mastitis were examined by light and electron microscopy at 6, 13, 20, and greater than 180 days after infection. With increasing time, there were increases in severity of granulomatous inflammation, number of endospores and sporangia, and ratio of degenerate to intact algae. Algae were found in macrophages but were not seen in neutrophils, epithelial cells, or myoepithelial cells. Macrophages containing algae were markedly enlarged, chiefly from reduplication of the Golgi complex and its associated vesicles. Intracellular algae were degenerate and consisted of intact cell wall profiles which contained membrane fragments but lacked nuclei and cytoplasmic organelles. Degenerate algae in vitro had thin cell walls and did not undergo internal lysis. Cell wall material of intracellular algae stained as an acidic, nonsulfated, carboxylated glycoprotein. These findings suggest that intracellular Prototheca zopfii degenerate by progressive lysis of internal organelles with persistence of cell wall glycans and that development of aberrant cell wall forms occurs as a defective response by host macrophages.
Analysis of paired sera from 48 wastewater workers and controls who reported gastrointestinal illness did not reveal any excess of seroconversions to Norwalk agent or to rotavirus. Inexperienced wastewater-exposed workers had higher levels of antibody to Norwalk agent than did experienced and control workers and those with high and medium aerosol exposure had higher titres than those in the low aerosol category. Analysis for Prototheca antibody titres was essentially negative.
A case of chronic meningitis caused by the achloric alga Prototheca wickerhamii is described, which has persisted for more than 6 years despite treatment with various antifungal agents. For the last year no treatment has been given, but the patient has no complaints.