Cytologic, histologic, microbiologic, and electron microscopic characterization of a canine Prototheca wickerhamii infection.

An adult dog was presented for chronic cough and a recent development of ulcerated, erythematous nares with nasal discharge. Cytology of enlarged peripheral lymph nodes revealed many intracellular and extracellular organisms. These round or rarely oval organisms measured approximately 5-9 µm in diameter and frequently contained several globular structures, ranging from deeply basophilic to magenta. A thin, clear halo was present. Smaller 1-2 µm, magenta forms were also observed. Fungal culture yielded small, wet, raised, irregularly shaped, white to pale tan colonies. Microbiologic staining of cultured material revealed features suggestive of algae. Histopathology of the lymph nodes revealed marked granulomatous inflammation with intralesional algal organisms suggestive of Prototheca. Electron microscopic findings were also consistent with protothecosis. Polymerase chain reaction, followed by direct DNA sequencing, identified the organism as Prototheca wickerhamii. A brief literature review discussing protothecosis in veterinary medicine is included.

Emergence of Fungal-Like Organisms: Prototheca.

The genus Prototheca consists of achlorophyllic algae that are ubiquitous in the environment and animal intestines. However, this organism has forfeited its photosynthetic ability and switched to parasitism. In 1894, Krüger described two microorganisms isolated in Germany from mucous flux of Tilia and Ulmus spp., namely Prototheca moriformis and P. zopfii. Based on their yeast-like colony morphology, Krüger classified these organisms as fungi. The genus is now included within the class Trebouxiophyceae, order Chlorellales, and family Chlorellaceae. Historically, protothecosis and infections caused by green algae have been studied in the field of medical mycology. Prototheca spp. have been found to colonize human skin, fingernails, the respiratory tract, and digestive system. Although human infection by Prototheca is considered rare, an increase in infections has been noted among immunosuppressed patients, those on corticosteroid treatment, or both. Moreover, the first human outbreak of protothecal algaemia and sepsis was recently reported in a tertiary care chemotherapy oncology unit in 2018. Prototheca is also a causative pathogen of bovine disease. Prototheca zopfii and P. blaschkeae are associated with bovine mastitis, which causes a reduction in milk production and secretion of thin, watery milk containing white flakes. Economic losses are incurred either directly via reduced milk production and premature culling of affected animals or indirectly as a result of treatment and veterinary care expenses. Thus, knowledge of this fungal-like pathogen is essential in human and veterinary medicine. In this mini-review, I briefly introduce human and animal protothecoses.

Canine ocular protothecosis: A review of 14 cases.

PURPOSE: To examine a series of 14 cases of canine ocular protothecosis from archived cases from the Comparative Ocular Pathology Laboratory of Wisconsin (COPLOW), and compare gross and histologic findings. METHODS: Archival records from COPLOW were searched for canine cases of ocular protothecosis. Fourteen cases that contained matching criteria were identified, and gross and histologic findings, and clinical records for each case were tabulated, examined, and compared (2001-2013). RESULTS: Of the 14 cases identified, six had evidence of systemic disease, as per clinical history. Two of the 14 cases had Prototheca identified via cytology of ocular fluid or retinal exudate; in the remainder of cases, Prototheca identified via cytology of ocular fluid or retinal exudate in the remaining 12 cases, Prototheca was identified upon histologic examination of the submitted globe(s). Presenting ocular clinical signs were variable and nonspecific. Duration of ocular clinical signs varied from days to months. Fundoscopically, white membranes or plaques were identified on or around the retina in five cases. Retinal detachment was identified in 13 of the 14 submitted globes on gross examination following fixation and sectioning. The predominant histologic finding was granulomatous chorioretinitis with retinal detachment, with variable numbers of Prototheca within the inflammatory infiltrate. CONCLUSIONS: Due to the nonspecific nature of the ocular signs, a diagnosis of protothecosis generally is not made until enucleation and histopathologic examination of the globe(s). Retinal detachment and blindness were common. Cytologic sampling of retinal plaques and exudate may provide a rapid way to identify Prototheca.

Comparative activity and mechanism of action of three types of bovine antimicrobial peptides against pathogenic Prototheca spp.

The yeast-like algae of the genus Prototheca are ubiquitous saprophytes causing infections in immunocompromised patients and granulomatous mastitis in cattle. Few available therapies and the rapid spread of resistant strains worldwide support the need for novel drugs against protothecosis. Host defence antimicrobial peptides inactivate a wide array of pathogens and are a rich source of leads, with the advantage of being largely unaffected by microbial resistance mechanisms. Three structurally diverse bovine peptides [BMAP-28, Bac5 and lingual antimicrobial peptide (LAP)] have thus been tested for their capacity to inactivate Prototheca spp. In minimum inhibitory concentration (MIC) assays, they were all effective in the micromolar range against clinical mastitis isolates as well as a Prototheca wickerhamii reference strain. BMAP-28 sterilized Prototheca cultures within 30-60 min at its MIC, induced cell permeabilization with near 100% release of cellular adenosine triphosphate and resulted in extensive surface blebbing and release of intracellular material as observed by scanning electron microscopy. Bac5 and LAP inactivated Prototheca following 3-6 h incubation at fourfold their MIC and did not result in detectable surface damage despite 70-90% killing, suggesting they act via non-lytic mechanisms. In circular dichroism studies, the conformation of BMAP-28, but not that of Bac5 or LAP, was affected by interaction with liposomes mimicking algal membranes. Our results indicate that BMAP-28, Bac5 and LAP kill Prototheca with distinct potencies, killing kinetics, and modes of action and may be appropriate for protothecal mastitis treatment. In addition, the ability of Bac5 and LAP to act via non-lytic mechanisms may be exploited for the development of target-selective drugs.

Molecular characterization of Polish Prototheca zopfii mastitis isolates and first isolation of Prototheca blaschkeae in Poland.

Bovine mastitits caused by the colorless, yeast-like alga Prototheca zopfii is a serious and complex condition that results in heavy economic losses in the dairy industry, both through a substantial reduction in milk production and culling of infected animals. Based on the 18S rDNA sequence analysis, genotype-specific PCR assays have recently been developed to differentiate within the species P. zopfii three distinct P. zopfii genotypes (1-3), of which P. zopfii genotype 3 has been considered a new species P. blaschkeae sp. nov. The purpose of this study was to employ the newly-devised molecular approach for the detection of the two P. zopfii genotypes and P. blaschkeae sp. nov. among bovine mastitis isolates from Poland. This study is the first to provide molecular characterization of Polish P. zopfii mastitis isolates. It also gives the first description of bovine mammary protothecosis due to P. blaschkeae in Poland, as evidenced by genotypical, microbiological, and electron microscopy findings.

Visualization of sporopollenin-containing pathogenic green micro-alga Prototheca wickerhamii by fluorescent in situ hybridization (FISH).

Fluorescent in situ hybridization (FISH) using taxon-specific, rRNA-targeted oligonucleotide probes is one of the most powerful tools for the rapid identification of harmful microorganisms. However, eukaryotic algal cells do not always allow FISH probes to permeate over their cell walls. Members of the pathogenic micro-algal genus Prototheca are characterized by their distinctive cell-wall component, sporopollenin, an extremely tough biopolymer that resists acid and alkaline hydrolysis, enzyme attack, and acetolysis. To our knowledge, there has been no report of the successful permeation by the oligonucleotide probes over the cell walls of unicellular green micro-algae, which contain sporopollenin. The DNA probes passed through the cell wall of Prototheca wickerhamii after treating the algal cells with cetyltrimethylammonium bromide (CTAB). Most cells in the middle logarithmic growth phase culture fluoresced when hybridized with the rRNA-targeted universal probe for eukaryotes, though individual cells included in this culture differed in the level of cell-wall vulnerability to attack by the polysaccharide-degrading enzyme, thus reflecting the different stages of the life cycle. This is the first report regarding the visualization of sporopollenin-containing, green micro-algal cells by FISH.

Diagnosis of clinical bovine mastitis by fine needle aspiration followed by staining and scanning electron microscopy in a Prototheca zopfii outbreak.

Biopsy by fine needle aspiration together with microbiological examination and scanning electron microscopy were evaluated in diagnosis of clinical bovine mastitis in a Prototheca zopfii outbreak. Fine needle aspiration was performed in 21 mammary quarters from ten Holstein cows presenting clinical mastitis caused by P. zopfii. The algae were previously identified in the microbiological examination of milk collected from these cows. Material aspirated from these 21 mammary glands was submitted to cytological staining (Gram, Giemsa and/or Shor staining). Fine needle aspiration enabled cytological identification of the algae in these 21 mammary glands, from which P. zopfii was isolated in the milk. Simultaneously, five mammary fragments collected by fine needle aspiration from these 21 mammary glands presenting clinical mastitis were also submitted to microbiological examination. P. zopfii was also isolated from these five fragments. Scanning electron microscopy technique also identified three of these five P zopfii strains isolated from mammary fragments collected by cytological aspiration. These results suggest that fine needle aspiration may be an alternative method for the diagnosis of clinical mastitis.

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.

[Cutaneous protothecosis treated with itraconazole]. / Protothécose cutanée traitée par itraconazole.

Protothecosis is a rare infection by algae without chloroplasts. We report a case of an immunocompetent patient who had visited countries of south-east Asia. The lesion was present on the elbow, and consisted of a granuloma studded with cells and sporangiae of Prototheca. A computerized image analysis revealed large variations in the size and shape of the organisms. The lesion was successfully treated with itraconazole.

The structural analysis of the mitochondrial SSUrRNA implies a close phylogenetic relationship between mitochondria from plants and from the heterotrophic alga Prototheca wickerhamii.

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.

Ultrastructure of Prototheca zopfii in bovine granulomatous mastitis.

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.

Metastatic protothecal retinitis in a dog. Electron microscopic observations.

Disseminated protothecosis, due to Prototheca wickerhamii, was present in a two-year-old female dog with a nine-month history of hemorrhagic colitis and diarrhea. Shortly thereafter, the dog developed "acute blindness" of the left eye. Euthanasia was done after medical therapy failed to control the disease. Histologically, the eye had multiple microabscesses and necrotic foci containing myriad protothecal organisms under the detached retina. Numerous organisms also were present in the mucosa and walls of the colon. The identification of P. wickerhamii was confirmed by the histologic appearance and immunofluorescent studies. The ultrastructural features of P. wickerhamii also were studied.

The effect of amphotericin B on the ultrastructure of Prototheca species.

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.

Rapid identification of Prototheca species by the API 20C system.

The conventional auxanographic method of testing for the assimilation of carbohydrates and alcohols by the various species of Prototheca requires at least 2 weeks of incubation at 25 to 30 degrees C before definitive results are obtained. Even though Prototheca spp., in culture as well as in fixed tissues, can be identified more rapidly by fluorescent-antibody techniques in which species-specific reagents are used, such diagnostic facilities and reagents are not available in most diagnostic laboratories. The API 20C clinical yeast identification system, a commercially available ready-to-use micromethod, was found to permit the definitive identification of P. stagnora, P. wickerhamii, and P. zopfii within 4 days.

Prototheca stagnora, an encapsulated organism.

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.