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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.
Protothecosis was described in many animals, with bovine mastitis being the main form. The increasing number of isolations of Prototheca spp. from bovine mastitis cases indicates the need of a detailed evaluation of this problem. Besides this, these algae do not respond to treatment with the antimicrobians most frequently applied, leading to elimination of the affected animals, as the best method to control the disease. In two dairy farms in the State of São Paulo, Brazil, a total of 155 lactating cows and 52 dry cows were examined. Milk samples were aseptically collected from lactating cows with clinical or subclinical mastitis. From dry cows, secretion samples from all quarters were collected. All samples were then taken to microbiological exams. At dairy farm 1, Prototheca spp. was isolated from 14.95% milk samples from lactating cows, (all of them cases of subclinical mastitis), and from 8.06% samples from dry cows. At dairy farm 2, Prototheca spp. was isolated from 5.1% milk samples and there were ten cases of clinical mastitis due to this agent. Although clinical mastitis has been considered the main form of occurrence of this pathology until now, it is important to consider Prototheca spp. as subclinical mastitis pathogen. Prototheca zopfii was the main specie isolated. In this study a high rate of intramammarian infections, as clinical or subclinical mastitis during lactation as well as in dry period, was observed, high lighting the importance of suitable diagnosis, so that control and preventive measures can be implemented to avoid dissemination of the agent.
In one cow all 4 quarters were infected with 2 x 10(8) germs of Prototheca zopfii each. 2 quarters of it were treated over 6 subsequent milking times with 20 ml Tetramisole hydrochloride (4 mg/kg body mass) in each case. Clinical symptoms diminished within 3-24 hours after the first application in comparison with the control quarters distinctly. Macroscopic alterations in the treated quarters could be found for a short time only, whereas they were evident in the control quarters for up to 2 days. The milk cell counts were raised til the 5. day p.i. The number of Prototheca excreted by treated quarters in each ml initial milk was reduced to 38% and in each ml end milk to 67% in comparison with the 2 control quarters. A more potent reduction of Prototheca can be expected after the application of Levamisole hydrochloride. At the future therapy applications after every milking time for at least 3 days with 25-40 ml of the preparations in each quarter are recommended. A total dose per animal and application time of 150 ml preparations (6 g Levamisole hydrochloride = 15 mg/kg body mass) must not be exceeded. Before Levamisole hydrochloride may be used for patients, susceptibility tests are necessary of the strains of Prototheca zopfii present and additional investigations for the reliability of the results hitherto obtained in one experimental Prototheca mastitis only.
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.
Prototheca spp. were culturally detected in 64 (28.9%) fecal samples of 221 pigs and in 3 (21.4%) fecal samples of 14 wild boars by using a selective medium, developed by PORE (1973). 25 (39.1%) of 64 Prototheca positive fecal samples of pigs contained Prototheca (P.) zopfii in monoculture, while in 39 fecal samples (60.9%) a mixed culture of P. zopfii and P. moriformis was found. All 3 fecal samples of wild boards contained a monoculture of P. zopfii. The results of this study permit the conclusion that pigs as well as wild boars can harbour and shed Prototheca spp. in variable frequency.
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.
Mastitis caused by Prototheca spp or Nocardia spp is considered to be difficult to treat. Both microorganisms are contaminants commonly found in soil. The occurrence of mastitis caused by these agents was studied in a particular dairy farm. In this herd, the animals were kept at pasture overnight and during daytime were brought to a pen where they were fed. This pen accumulated mud and faeces, particularly in the rainy season. During milking, pre-dipping of the teats was performed with an iodide solution, but they were not washed, so a layer of soil and faeces remained which may have contaminated the milking equipment. The herd comprised 91 lactating animals and 47 dry cows. For microbiological examination, 107 milk samples were collected from lactating cows and 186 samples of mammary secretions from the dry cows. Prototheca spp were isolated from 14.55% of the milk samples and Nocardia spp from 4.55%. Prototheca spp were isolated from 8.06% of the secretion samples from dry cows and Nocardia spp were isolated from 2.15% samples. The high occurrence of mastitis due to these environmental agents reflects the problem of keeping animals in muddy pastures and pens, and the defective pre-milking hygiene for the teats.
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.
Prototheca sp., a colourless algae, is quite common in dairy environments, particularly in wet areas contaminated with manure. The main purpose of this paper is to describe an outbreak of clinical bovine mastitis in an 86-cow dairy herd in the State of Säo Paulo, Brazil. Prototheca sp., an achlorophyllous algae, were isolated on blood agar (incubated for 24 h at 37 degrees C) from 11 quarters of seven lactating Holstein cows, and from one quarter of a cow at the end of the dry period. Treatments were applied, but there was only a microbiological cure, not a functional one. Diagnosis of Prototheca sp. in any of the cows in the herd indicates a herd problem. Infected animals usually have markedly reduced milk production and granulomatous changes often occur in the mammary gland. All sources of contact between the teat ends and drainage water or damp areas should be corrected. An all-out effort for strict sanitation, including during milking, should be made so that the teat ends will not become contaminated.
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)
Preparations with 4 percent Tetramisole or Levamisole hydrochloride were compatible in the mammary gland after application up to 40 ml/quarter (4 mg/kg body mass) for up to 6 milking times. 100 ml of the preparations were also clinically compatible in a single quarter (10 mg/kg body mass) on several days. After intramammary application of 30 ml/quarter (3 mg/kg body mass) Prototheca suppressing values in milk serum were evident for at least 6 hours. One application of 200 ml in one quarter has caused Prototheca suppressing values for 36 hours in milk serum up to dilution 1:4. Nilverm given to cattle in high dosage orally did not lead to Prototheca suppressing values in milk sera.