A rapid real-time PCR/DNA resolution melting method to identify Prototheca species

Aim: The study describes the development of a simple and rapid tool to identify yeast‐like microalgae belonging to the genus Prototheca. Methods and Results: The method, based on two‐step Real Time PCR reaction followed by DNA Resolution Melting Analysis (qPCR/RMA), has been developed using reference strains belonging to both pathogenic (P. zopfii genotype 2, P. wickerhamii and P. blaschkeae) and non‐pathogenic species (P. zopfii genotype 1, P. stagnora and P. ulmea). In order to validate the method, seventy recently isolated Prototheca strains were thus tested in parallel with both the first qPCR/RMA and the conventional genotype‐specific PCR assay: they were classified as P. zopfii genotype 1, P. zopfii genotype 2 and P. blaschkeae, with a perfect accordance between the two above methodologies. Furthermore, we used the second qPCR/RMA to identify the other species (P. stagnora, P. ulmea and P. wickerhamii), which cannot be discriminated by conventional PCR assay. Conclusions: The assay two‐step Real Time PCR is accurate, robust, cost‐effective and faster than auxonographical, biochemical or conventional molecular biology methods. Significance and Impact of the Study: the rapid and high throughout two‐step qPCR/RMA tool can be usefully used for the identification of clinical and environmental Prototheca species into the framework of the diagnosis of animal (e.g. bovine mastitis) or human protothecosis.     

MOLECULAR PHYLOGENY AND PHENOTYPIC VARIATION IN THE HETEROTROPHIC GREEN ALGAL GENUS PROTOTHECA (TREBOUXIOPHYCEAE,CHLOROPHYTA)1

Species of the heterotrophic green microalgal genus Prototheca and related taxa were phylogenetically analyzed based on the nuclear small subunit (SSU) and the 5′ end of the large subunit (LSU) rRNA gene (rDNA) sequences. We propose restricting the genus Prototheca to the four species: P. moriformis Krüger, P. stagnora (Cooke) Pore, P. ulmea Pore, and P. zopfii Krüger. The main diagnostic feature of these taxa is the absence of growth on trehalose.Of these, it was suggested that P. moriformis should be merged into P. zopfii; P. moriformis and three varieties of P. zopfii constituted a paraphyletic assemblage with estimated short evolutionary distances. The trehalose‐assimilating strains (Prototheca wickerhamii Tubaki et Soneda strains and Auxenochlorella protothecoides (Krüger) Kalina et Pun?ochá?ová SAG 211‐7a), together with an invertebrate pathogen Helicosporidium sp., diverged before the radiation of the four species of Prototheca in the SSU rDNA and composite (SSU rDNA plus LSU rDNA) analyses. Comparison between the results from physiological data in this work (fermentative pattern) and those described earlier (growth requirements) lead us to propose a hypothesis that the phenotypic variation, which did not represent diagnostic characters for species delimitation, may reflect the history of genetic diversification within the genus Prototheca as inferred from rDNA sequence characters.     

Prototheca associated with banana

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.     

Identification of a unicellular,non-pigmented alga that mediates growth inhibition in anuran tadpoles: a new species of the genus Prototheca (Chlorophyceae: Chlorococcales)

A non-pigmented, unicellular alga isolated from the faeces of British anuran tadpoles and which is associated with growth inhibition in these tadpoles, was described and identified using cytological, ultrastructural, nutrient assimilation and immunological studies. The alga possessed all the distinctive morphological features of the genus Prototheca, it grew weakly on Prototheca Isolation Medium (PIM), it required thiamine for continued growth and replication, and it could assimilate the five major substrates used to speciate the protothecans. All of these characteristics, together with previous nucleic acid hybridisation studies, indicated that the microorganism belonged to the genus Prototheca. There are currently five species recognised as valid (Pore, 1985 & 1986): Prototheca zopfii Kruger, 1884, P. wickerhamii Tubaki & Soneda, 1959, P. moriformis Kruger, 1884, P. stagnora Cooke, 1968 and P. ulmea Pore, 1986.The immunology showed that the new species was related to two of the protothecans, but overall it showed that the alga was antigenically distinct from the other protothecans tested in the immunoassay. This, together with its inability to grow strongly on PIM, its ability to assimilate a wide rage of carbon substrates and its ability to mediate growth inhibition in anuran tadpoles, indicated a new species of Prototheca. We therefore propose the name Prototheca richardsi sp. n.     

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.     

Physiological and biochemical contributions to the taxonomy of the genus Prototheca

Prototheca zopfii (12 strains) is able to use glucose, fructose, propanol, glycerol, and acetate as sources of carbon for growth. One of the strains is biochemically (utilization also of galactose and mannose), and two strains are morphologically slightly different.Two strains can be identified as P. wikerhamii. They exhibit good growth with glucose, fructose, galactose, trehalose, propanol, glycerol, acetate, and glutamate as sources of carbon. P. spec. 263-2 grows only with glucose and acatate. P. zopfii and P. wickerhamii are able to use urea, glycine, and glutamate as sources of nitrogen. P. spec. 263-2, on the other hand, cannot utilize these organic nitrogen compounds for growth.Four strains of Chlorella protothecoides are able to use glucose, fructose, galactose, and acetate as sources of carbon for growth in the dark. Three of them utilize also mannose, trehalose, and glutamate. Two strains can grow with glycerol, and one is able to use lactose. — Urea and glycine can serve as sources of nitrogen for the four strains of C. protothecoides. Glutamate supports growth of three strains, and one strain is able to use nicotinamide.     

Prototheca zopfii: Natural,transient, occurrence in pigs and rats

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.     

Human Cutaneous Protothecosis: A Case Report and Review of Cases from Mainland China,Hong Kong,and Taiwan

We present a case of cutaneous protothecosis caused by Prototheca wickerhamii infection. The patient was a 72-year-old man with hypoalbuminemia. He responded well to fluconazole treatment. We reviewed this case along with 17 other cases of cutaneous protothecosis reported from mainland China, Hong Kong, and Taiwan. Of the 18 cases, 7 each occurred in mainland China and Taiwan, and 4 occurred in Hong Kong. Thirteen cases were caused by P. wickerhamii (72.2%), and three were caused by P. zopfii (16.7%); in two cases, the species was not identified (11.1%). In all, 9 (50%) patients were immunocompromised, and 10 (55.5%) patients denied having a history of trauma. All patients presented with polymorphic skin lesions, and erythematous papules, plaques, or nodules was the most common presentation (15/18, 83.3%). Genotyping was performed in five cases, mostly by means of small subunit ribosomal DNA amplification (four cases). Susceptibility tests (6 patients) showed that P. wickerhamii was sensitive to amphotericin B and voriconazole but resistant to fluconazole or itraconazole. Treatment succeeded in 15 (83.3%) patients and failed in 3 (16.7%). Our data indicate that the number of cutaneous protothecosis cases is underestimated in China, and the skin lesions have some diagnostic value.     

Cinco cepas de Prototheca de origen humano

Resumen Se han aislado cinco cepas dePrototheca de muestras de origen humano: raspado vulvar, tinea corporis, secreción de oído, plieques de la mano, onicopatía inespecífica. Se estudian los caracteres morfológicos (propiedades asimilativas, termofilia) de estas cepas. Dos de ellas pertenecen a la especiePrototheca zopfii. Dos a la especieP. wickerhamii y la quinta es unaP. trispora. El autor hace resalter la frecuencia cada vez mayor con que se viene aislando este género de algas incoloras en el material patológico y la necesidad de su reconocimiento ya que son fácilmente confundibles con las levaduras.

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FivePrototheca strains of human origin are isolated. Morphological and physiological characters are studied. Two strains belong to the speciesPrototheca zophii, two to the speciesP. wickerhamii and the other one toProtheca trispora. The author emphasizes the frequency, with which this genus of chloric algae are lately reported, and the importance of their identification easily confused with that of the yeasts.Ultimamente hemos venido encontrando cada vez con mayor frecuencia en el material clínico, ejemplares del género Prototheca. La ubicación taxonómica de este género es aún dudosa; se le ha considerado un grupo intermedio entre algas incoloras y hongos (Tubaki, 1959). Nuestras 5 cepas han sido encontradas en un lapso de cuatro años, de un total de 320 muestras.

     

A Case of <Emphasis Type="Italic">Prototheca zopfii</Emphasis> Genotype 1 Infection in a Dog (<Emphasis Type="Italic">Canis lupus familiaris</Emphasis>)

Protothecosis is a rare disease caused by environmental algae of the genus Prototheca. These are saprophytic, non-photosynthetic, aerobic, colorless algae that belong to the Chlorellaceae family. Seven different species have been described. Prototheca zopfii genotype 2 and P. wickerhamii are most commonly involved in pathogenic infections in humans and animals. The objective of this work is to describe, for the first time, a case of protothecosis caused by P. zopfii genotype 1 in a dog. The dog, a 4-year-old mix bred male, was presented to a veterinary clinic in Montevideo, Uruguay, with multiple skin nodules, one of which was excised by surgical biopsy. The sample was examined histologically and processed by PCR, DNA sequencing, and restriction fragments length polymorphisms for the detection and genotyping of P. zopfii. In addition, transmission electron microscopy and scanning electron microscopy were performed. Histology showed severe ulcerative granulomatous dermatitis and panniculitis with myriads of pleomorphic algae. Algal cells were 4–17 µm in size, with an amphophilic, 2–4-µm-thick wall frequently surrounded by a clear halo, contained flocculant material and a deeply basophilic nucleus, and internal septae with daughter cells (endospores) consistent with endosporulation. Ultrastructurally, algal cells/endospores at different stages of development were found within parasitophorous vacuoles in macrophages. Prototheca zopfii genotype 1 was identified by molecular testing, confirming the etiologic diagnosis of protothecosis.     

Direct fermentation of cellodextrins to ethanol byCandida wickerhamii andC. Lusitaniae

Summary Production of ethanol from cellodextrins, as large as cellohexose, byCandida lusitaniae andC. wickerhamii was studied.C. lusitaniae fermented only glucose and cellobiose, whereasC. wickerhamii efficiently fermented cellodextrins. Maximum ethanol yields of 29.2 g/liter from 54 g/liter cellodextrins were achieved byC. wickerhamii in 3–4 days.     

A novel DNA extraction and duplex polymerase chain reaction assay for the rapid detection of Prototheca zopfii genotype 2 in milk

Aims: To develop a PCR‐based assay to detect Prototheca zopfii (P. zopfii) and its mastitis‐related subtype (genotype 2) directly from milk samples. Methods and Results: The DNA extraction method herein is based on the lysing properties of chemical agents, mechanical grinding and DNA‐binding properties of silica particles; this method was developed to rapidly extract DNA directly from P. zopfii in bovine milk. Two pairs of primers specific for P. zopfii and genotype 2 were used in the duplex PCR, and a sensitivity test showed that the detection level was 5 × 10² colony‐forming units (CFU) ml^?1 for P. zopfii and 5 × 10³ CFU ml^?1 for genotype 2. Furthermore, a practical survey of 23 milk samples showed that the assay produced results that were in accordance with those obtained by the conventional microbiology method. Conclusions: The DNA extraction method is effective in isolating sufficient quantities of DNA from P. zopfii in milk for PCR analysis. The PCR assay is economical, sensitive and more rapid than the conventional culture method. Significance and Impact of the Study: The assay could be used as an alternative method for the rapid the detection of bovine mastitis resulting from P. zopfii genotype 2.     

Characterization of <Emphasis Type="Italic">Prototheca zopfii</Emphasis> Associated with Outbreak of Bovine Clinical Mastitis in Herd of Beijing,China

Prototheca zopfii (P. zopfii) has become an important cause of bovine mastitis in many countries. In the present study, to better understand the occurrence of one clinical mastitis (CM) outbreak due to P. zopfii, the molecular characterization and resistance patterns of the microalga were described. P. zopfii strains were isolated from 17 of 23 quarters, which suffered CM in the outbreak, and 7 of 46 CM recovered quarters before the outbreak, as well as 2 of 75 environmental samples in the dairy farm. All strains were identified as genotype 2 by genotype-specific PCR analysis. Results of in vitro antimicrobial and antifungal susceptibility tests indicated that these strains were resistant to majority of tested drugs, with the only exception of amphotericin B, nystatin, streptomycin, gentamicin, and amikacin. This is the first report about CM outbreak caused by P. zopfii in China. These data suggest that P. zopfii may represent a serious risk in the studied herd, and this microalga could be an important potential pathogen causing mastitis in dairy herds of Beijing.     

Evolution of fragmented mitochondrial ribosomal RNA genes inChlamydomonas

The fragmented mitochondrial ribosomal RNAs (rRNAs) of the green algaeChlamydomonas eugametos andChlamydomonas reinhardtii are discontinuously encoded in subgenic modules that are scrambled in order and interspersed with protein coding and tRNA genes. The mitochondrial rRNA genes of these two algae differ, however, in both the distribution and organization of rRNA coding information within their respective genomes. The objectives of this study were (1) to examine the phylogenetic relationships between the mitochondrial rRNA gene sequences ofC. eugametos andC. reinhardtii and those of the conventional mitochondrial rRNA genes of the green alga,Prototheca wickerhamii, and land plants and (2) to attempt to deduce the evolutionary pathways that gave rise to the unusual mitochondrial rRNA gene structures in the genusChlamydomonas. Although phylogenetic analysis revealed an affiliation between the mitochondrial rRNA gene sequences of the twoChlamydomonas taxa to the exclusion of all other mitochondrial rRNA gene sequences tested, no specific affiliation was noted between theChlamydomonas sequences andP. wickerhamii or land plants. Calculations of the minimal number of transpositions required to convert hypothetical ancestral rRNA gene organizations to the arrangements observed forC. eugametos andC. reinhardtii mitochondrial rRNA genes, as well as a limited survey of the size of mitochondrial rRNAs in other members of the genus, lead us to propose that the last common ancestor ofChlamydomonas algae contained fragmented mitochondrial rRNA genes that were nearly co-linear with conventional rRNA genes.     

Effect of Prototheca zopfii on neutrophil function from bovine milk

This study was carried to investigate neutrophil function in the presence of Prototheca zopfii. For this purpose, bovine milk neutrophils were incubated in the absence (control) of and presence of P. zopfii, and then they were examined hydrogen peroxide (H₂O₂) production, antioxidant enzyme activities, and phagocytic capacity. Milk was collected from negative “California Mastitis Test” (CMT) quarter from three lactating Holstein cows after induction of leukocytosis with an intramammary infusion of oyster glycogen. H₂O₂ production was measured using the phenol red method. Catalase activity was measured following H₂O₂ reduction at 240 nm and the activity of glutathione reductase was determined by measuring the rate of NADPH oxidation at 340 nm. P. zopfii death was assessed by fluorescent microscopy using acridine orange assay and by colony forming units (CFUs). Comparisons between the groups were initially performed by analysis of variance (ANOVA). Significant differences were then compared using Tukey’s test with a significance coefficient of 0.05. Hydrogen peroxide production, catalase and glutathione reductase activities by neutrophils incubated in presence of P. zopfii were stimulated five times, 21% and 27% respectively, compared to the unstimulated-neutrophils. Neutrophils did not affect P. zopfii death as shown by microscopy and CFUs. These observations led to the conclusion that the P. zopfii promote a high increase of H₂O₂ production by neutrophils from bovine milk during algae exposition accompanied by increase of antioxidant enzyme activities; however, this process did not affect P. zopfii death.     

Preparation and regeneration of mycelial protoplasts ofPleurotus florida andP. ostreatus

A method for isolation of higher frequency of regenerated protoplast fromPleurotus florida andP. ostreatus is reported.     

Effect of Different Heat Treatments and Disinfectants on the Survival of <Emphasis Type="Italic">Prototheca zopfii</Emphasis>

Bovine mastitis caused by the yeast-like alga Prototheca zopfii represents a serious veterinary problem and may result in heavy economic losses to particular dairy farms. The purpose of this study was to evaluate the survival of 50 isolates of P. zopfii in milk subjected to different heat treatments and the survival of further 106 P. zopfii isolates after exposure to three classes of teat disinfectants: iodine (Dipal), quaternary ammonium compounds (Teat), and dodecylbenzenesulphonic acid (Blu-gard). Of the 50 isolates tested for thermal tolerance, 29 (58%) survived heat treatment at 62°C for 30 s and 13 (26% of all isolates) of those survived after heat treatment at 72°C for 15 s. None of the 106 isolates were able to withstand the in-use concentrations of the three disinfectants tested. The highest disinfectant concentrations that permitted survival of at least one isolate were dilutions: 1:1,000 for Dipal (survival rate of 52.8–57.5%), 1:100 for Teat (88.7–90.6%), and 1:10 for Blu-gard (100%). No differences in the survival rates of P. zopfii were observed with respect to the duration of exposure to disinfectant. The results of this study support the previous findings that P. zopfii may resist high-temperature treatments, including that applied in the high-temperature, short-time (HTST) pasteurization process. The obtained data also demonstrate the efficacy of the three classes of teat disinfectants against P. zopfii, with the efficacy of iodine being most pronounced. The study emphasizes the necessity of using higher temperatures in the pasteurization of raw milk to kill the Prototheca algae, as well as the particular suitability of the iodine for the control procedures of protothecal mastitis.     

Two‐dimensional proteome reference map of Prototheca zopfii revealed reduced metabolism and enhanced signal transduction as adaptation to an infectious life style

Biochemical, serological, and genetic analyses have identified two genotypes of Prototheca zopfii, a unicellular microalga belonging to the family Chlorellaceae. The P. zopfii genotype 1, abundantly present in cow barns and environment, remains nonpathogenic, while P. zopfii genotype 2 has been isolated from cows with bovine mastitis. The present study was carried out to identify the protein expression level difference between the pathogenic and nonpathogenic strains of P. zopfii. A total of 782 protein spots were observed on the 2D fluorescence difference gel electrophoresis (2D DIGE) gels among which 63 and 44 proteins were identified to be overexpressed in genotypes 1 and 2, respectively. The limited number of protein entries specific for Prototheca in public repositories resulted mainly in the identification of proteins described in other algae, microorganisms, or plants. Gene ontology (GO) analysis indicated reduced carbohydrate metabolism in genotype 1, while genotype 2 displayed enhanced DNA binding, kinase activity, and signal transduction. These effects point to metabolic and signaling adaptations in the pathogenic strain and provide insights into the evolution of otherwise highly similar strains. All MS data have been deposited in the ProteomeXchange with identifier PXD000126.     

Spatial distribution of cpDNA and mtDNA haplotypes in a hybrid zone betweenPinus pumila andP. parviflora var.pentaphylla (Pinaceae)

Pinus species exhibit paternal chloroplast inheritance and maternal mitochondrial inheritance. This independent inheritance of two cytoplasmic genomes provides an exceptional environment for discriminating female (seeds) and male (pollen) components of gene flow across hybridizing species. We obtained mitochondrial genetic markers diagnostic toP. parviflora var.pentaphylla andP. pumila by PCR amplification of the intron ofnad1 on mtDNA, and examined the spatial-distribution pattern of the mtDNA haplotypes in a hybrid zone betweenP. parviflora var.pentaphylla andP. pumila in the Tanigawa Mountains of Japan. These data, in conjunction with previous information on cpDNA haplotypes and needle morphology, revealed contrastive patterns of introgression of two cytoplasmic genomes. CpDNA introgression has occurred uni-directionally fromP. parviflora var.pentaphylla toP. pumila. Conversely, mtDNA introgression has occurred in the opposite direction, fromP. pumila toP. parviflora var.pentaphylla. Levels of introgression are roughly equivalent for cpDNA and mtDNA. The contrastive spatial distribution pattern of cpDNA and mtDNA haplotypes could be caused by differential movement of seeds and pollen for interspecific genetic exchange.     

Uncommon Fungi Isolated from Diabetic Patients Toenails With or Without Visible Onychomycoses

Kodamaea ohmeri and Prototheca wickerhamii are rare pathogens for humans, and even more rare as cause of onychomycosis. This work reports the second case of onychomycosis by K. ohmeri and the fourth of onycoprotothecosis; it was made in public health institutions in the Hidalgo State, Mexico, studying 261 diabetic patients during 2005 and 2006. Kodamaea ohmeri was isolated from toenails of a 51-year-old female patient, and P. wickerhamii from three female patients of 48, 49, and 61 years old, respectively, all of them with type 2 diabetes mellitus (DM 2). Identifications were done by standard microbiological methods and a commercial system. Only one patient infected with P. wickerhamii showed mixed infection with dermatophytes. Out of the total studied DM 2 patients, 1.15% presented onycoprotothecosis and 0.38% onychomycosis by K. ohmeri, high percentages if it is considered that few cases have been reported of K. ohmeri and P. wickerhamii as onychomycosis causal agents.