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A simple new test to differentiate Prototheca wickerhamii from Prototheca zopfii by determining susceptibility to clotrimazole is described. A 50-micrograms clotrimazole disk provides a rapid and reliable means of distinguishing P. wickerhamii from P. zopfii.
Twenty isolates of Prototheca filamenta, Prototheca moriformis, Prototheca stagnora, Prototheca wickerhamii, and Prototheca zopfii were tested for in vitro susceptibility to five commonly used antifungal agents: amphotericin B, 5-fluorocytosine, griseofulvin, miconazole, and nystatin. The results revealed resistance to griseofulvin of all the Prototheca isolates tested and an inhibitory effect on P. filamenta by high 5-fluorocytosine concentrations (minimal inhibitory concentration [MIC] = 12.5 to 100 mug/ml; minimal fungicidal or algacidal concentration [MFC/MAC] = 50 to 100 mug/ml). P. filamenta isolates were also susceptible to miconazole (MIC = 0.1 to 0.5 mug/ml, MFC/MAC = 0.5 to 1 mug/ml); isolates of the other Prototheca species varied in regard to miconazole activity from susceptible to resistant (MIC = 1 - >100 mug/ml, MFC/MAC = 5 - >100 mug/ml). The Prototheca isolates revealed an in vitro susceptibility to the polyene antifungal agents, amphotericin B, and nystatin (MIC = 0.09 to 3.12 mug/ml and 0.19 to 12.5 mug/ml, respectively; MFC/MAC = 0.19 to 25 mug/ml and 0.75 to 25 mug/ml, respectively).
One hundred strains of Prototheca zopfii, Prototheca wickerhamii, Prototheca moriformis, Prototheca stagnora, and Prototheca ulmnea; five strains of Chlorella protothecoides; and two strains of Candida albicans were obtained from a number of different clinical and environmental sources and were tested for their in vitro susceptibility to the antibacterial agent gentamicin. All Prototheca strains were susceptible to gentamicin at concentrations between 0.3 and 0.9 micrograms/ml. A modified macrobroth dilution MIC assay with a colorimeter and a microbroth dilution assay with a 96-well plate reader were the two methods used to determine the MICs.
A source of Prototheca spp causing mastitis in a herd of 263 milking cows in North Carolina was investigated. Of 38 samples from the dairy environment, 18 (47%) contained Prototheca spp. Isolation sites included cattle drinking water; a feed trough; mud, dirt, and excreted feces from a dirt lounging area; water, sludge, mud, and vegetation from a creek in the lounging area; and the floor of a freestall barn. Samples were collected from 5 additional dairies, including one dairy with and 4 dairies without a history of protothecal mastitis. Prototheca spp were isolated from 48 (25%) of 190 samples from various sites on all 6 dairies. Isolates were P zopfii (45; 94%) and P wickerhamii (3; 6%). Isolation frequency ranged from 4 to 47% of samples/dairy. There was no apparent difference in the isolation frequency of Prototheca spp from samples from dairies with or without a history of protothecal mastitis. Sites characterized by wetness and the presence of organic matter most commonly yielded Prototheca spp. Because Prototheca spp appear to be common in the dairy environment, factors in addition to presence in the environment may be important in development of protothecal mastitis.
Studies were carried out to develop fluorescent antibody reagents for the identification of the Prototheca species and for their differentiation from morphologically similar fungi of various genera in formalin-fixed tissues. Antisera against representative isolates of P. filamenta, P. moriformis, P. stagnora, P. wickerhamii, and P. zopfii were produced in rabbits. Antiglobulins, labeled with fluorescein-isothiocyanate that intensely stained most cells of the homologous species, were selected for use as potential diagnostic reagents. By adsorbing the conjugates with selected heterologous cross-staining protothecae, reagents that were both sensitive and specific were obtained. Evaluation of the adsorbed conjugates with sections of tissue infected with protothecae, sections of tissue infected with morphologically similar fungi, and cultures of protothecae showed that these reagents are useful for the rapid and reliable identification of the Prototheca species.
Prototheca zopfii was isolated from cow-composite milk and bulk tank milk by a new selective Prototheca enrichment method. Repeated testing of cow-composite milk from individual cows resulted in P. zopfii isolation data indicating a strong statistical correlation of P. zopfii with specific cows. Prototheca sp. were isolated from the milk of 31 of 79 cows in a single herd, and contamination was discounted as the source. Prototheca sp. were also recovered from 28 of 787 bulk tank milk samples from the Eastern U.S.A., and 22 of 69 temperature control milk samples from pooled dairy farm milk from delivery trucks.
A new staining solution named "PHOL" is described for studying the morphology of clinical and environmental isolates of fungi and Prototheca. In total 69 isolates were used for assessing the efficacy of this newly formulated stain. The solution has shown good ability to stain all 62 isolates of fungi and 7 of Prototheca, and thus helped in the identification of the organisms. The new solution has the potential to stain the young as well as old isolates of fungi and Prototheca. It is emphasized that a wider application of this newly introduced inexpensive and safe staining solution will help the mycologists to study the morphological characteristics of yeast, molds, and Prototheca obtained from man, animals and environment.
Biopsy of a wound infection of the palmar fascia in a young diabetic woman revealed characteristic periodic acid-Schiff-positive Prototheca species cells with a rosette configuration and internal septation. Prototheca wickerhamii was cultured repeatedly from the wound drainage and the biopsy tissue. Several diagnostic features distinguishing Prototheca species, saprophytic algae, from yeasts are: the formation of endospores by mitosis; greater variation in cell size (2 to 15 mum); the presence of cytoplasmic granules, particularly in old cultures; and the absence of budding forms and pseudomycelia. The organism was resistant to 5-fluorocytosine and the minimal inhibitory concentration of amphotericin B was 12.5 mug/ml. With the exception of the tetracycline group, all other 16 antibacterial agents tested appeared completely ineffective in vitro. A synergism between amphotericin B and tetracycline was clearly demonstrated by the use of the checkerboard method. Infection by Prototheca species may be more common than presently realized due to the common expedient of identifying yeast-like isolates as "yeast--not Candida albicans."
Evidence that algae are pathogens was provided by the results of electron microscopic studies of tissues from five cattle and sheep suspected of having green algal infections. Chloroplasts were demonstrated in the algae in each case. Prototheca organisms, considered by some to be achloric mutants of green algae, are causative agents of disease in man and animals and may appear morphologically similar to green algae in tissue. However, electron microscopy showed that chloroplasts were absent in these organisms. Light microscopy revealed not only similarities in size, shape, and mode of reproduction, but also a striking difference between the Prototheca organisms and green algae. Unlike Prototheca, the green algae contained abundant cytoplasmic starch granules that were strongly positive by several staining procedures; these granules, which were PAS-negative following diastase digestion, provide a means of differentiating green algae from Prototheca cells in tissue.
A differential test was made between genus Candida and genus Prototheca using a new and very simple differential test. A total of 59 strains of Candida and 78 strains of Prototheca were used. The basis of the test was the differential use of a disc carrying 60 mcg of Ribostamycin to which all the Candida were resistant and the Prototheca inhibited.
The proof of a chlorophyllous alga of the genus Prototheca from the blood of a tuberculous patient with brain damage is described. The pathogenetic importance of Protothecae in the field of human medicine is discussed. Although representatives of the genus Protothecae up to now are regarded as extraordinarily infrequent commensals, nevertheless occasionally their proof not only on the surface of the skin but also from the blood or the inner organs must be taken into account.
Prototheca wickerhamii, an achlorophyllous algae, was previously found to induce massive epithelioid cell granulomas in the skin of mice. By means of light microscopy, examination was made of the histological reactions involved in epithelioid cell granulomas induced by intradermal and/or subcutaneous inoculation of Prototheca wickerhamii in BALB/c and ICR mice. Six BALB/c mice showed granuloma nodules while only three of six ICR mice did so. Based on the results of the present and previous studies, BALB/c mice may be considered a strain particularly vulnerable to contracting epithelioid cell granuloma and ICR mice, a resistant strain. In very early lesions at one week following initial prototheca inoculation, cellular infiltration with varying numbers of polymorphonuclear leukocytes, lymphocytes and some macrophages was observed throughout the dermis and subcutaneous fat tissue. In early lesions at one to two months after inoculation, focal granulomas composed of histiocytic cells and/or macrophages were observed. Mast cells were occasionally present among the histiocytic cell infiltrates. In the granulomatous lesions at two to three months, scattered eosinophils and some lymphocytes were seen. Central necrosis, with numerous neutrophils and many endospores surrounded by the granuloma, was often observed. In late stage lesions at six months, massive lymphocyte and plasma cell infiltration surrounding and/or intervening between vacuolated epithelioid cell clusters was evident. Histological reactions in epithelioid cell granuloma and the ultimate course of this disease can be staged from the histological point of view as follows: 1) diffuse inflammation, 2) cell proliferation leading to epithelioid cell formation, 3) hypertrophy of epithelioid cells with consequent formation of cell aggregates and/or organized granuloma and 4) degeneration of granuloma.(ABSTRACT TRUNCATED AT 250 WORDS)
The gene for the mitochondrial small subunit rRNA (SSUrRNA) from the heterotrophic alga Prototheca wickerhamii has been isolated from a gene library of extranuclear DNA. Sequence and structural analyses allow the determination of a secondary structure model for this rRNA. In addition, several sequence motifs are present which are typically found in SSUrRNAs of various mitochondrial origins. Unexpectedly, the Prototheca RNA sequence has more features in common with mitochondrial SSUrRNAs from plants than with that from the green alga Chlamydomonas reinhardtii. The phylogenetic relationship between mitochondria from plants and algae is discussed.
Prototheca stagnora was found to be a habitant of older harvested banana (Musa sapientum) and plantain (M. paradisiaca) stumps while P. wickerhamii colonized fresh Musa sp. stumps and flower bract water of Heliconia sp. While Prototheca sp. were known to habituate woody plants, this is the first evidence that herbaceous plants also serve as habitats.
Prototheca zopfii and prototheca wickerhamii stains 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.
Domestic swine faeces and fresh faeces from trapped barnyard rats were heavily contaminated with Prototheca zopfii, a cause of dairy cow mastitis. When the pigs and rats were maintained on Prototheca-free diets, the transient intestinal population of P. zopfii decreased precipitously and disappeared. When combined with the information that other farm animals excrete P. zopfii, it was concluded that contaminated animal feed may be the source of large numbers of P. zopfii in the farm environment. We found P. zopfii in wet spoiled feed. Rats are logical vectors for contamination of feed.
Prototheca zopfii was isolated from a patient with olecranon bursitis. Olecranon bursitis caused by Prototheca is a distinct clinical entity. Recognition of this infection was made by observing characteristic organisms in tissue and subsequent isolation of the organism.
Fifty-two cultures of Prototheca spp. isolated from water and 7 isolates received from a culture collection were tested for their ability to assimilate carbon and nitrogen sources. Based upon these findings and on micromorphological features of the isolates a rapid method allowing differentiation of Prototheca spp. in culture is presented.