Prototheca Species Research Articles

Perspectives and Possibilities for New Antimicrobial Agents in the Treatment and Control of Mastitis Induced by Algae of the Genus Prototheca spp.: A Review.

Innovative approaches in nanotechnology provide a potentially promising alternative to untreatable cases of mastitis caused by genus Prototheca spp. algae infections. Drying of the teats of the affected animals or culling are typically the outcomes of mastitis in dairy cattle caused by these pathogens. A major issue in both veterinary medicine and animal breeding is the Prototheca species' widespread resistance to the current methods of managing infections and the available drugs, including antibiotics. Commercial antifungal preparations are also ineffective. Nanotechnology, an emerging discipline, has the potential to create an effective alternative treatment for protothecal mastitis. The aim of the paper is to combine the literature data on the use of nanotechnology in the control of mastitis, taking into account data on combating mastitis caused by Prototheca spp. infections. The databases employed were PubMed, Google Scholar, and Scopus, focusing on literature from the last 20 years to ensure relevance and currency. Studies conducted in vitro have demonstrated that nanomaterials have significant biocidal activity against mastitis infections of different etiologies. Analyzed research papers show that (NPs), such as AgNPs, CuNPs, AuNPs, etc., may not negatively impact various cell lines and may be effective agents in reducing the pathogens' viability. However, it is also critical to assess the risks involved in using nanomaterials.

In vitro susceptibility testing of phytochemicals from essential oils against Prototheca species.

Phytochemicals isolated from essential oils are effective alternatives for inhibiting microbial pathogens. Bovine protothecal mastitis is the cause of a reduction in milk production and the secretion of thin, watery milk with white flakes. In the present study, we performed in vitro susceptibility testing of the phytochemicals carvacrol, citral, and thymol in Prototheca strains isolated from cases of protothecosis in small animals and cow feces. The susceptibility of the algae to carvacrol, citral, and thymol was assessed using the modified CLSI M27-A3 broth microdilution method. The ranges of the minimum inhibitory concentrations (MIC%) of the phytochemicals in all isolates were 0.03% to 0.125% for carvacrol, 0.03% to 0.25% for citral, and 0.06% to 0.25% for thymol. Based on these results, carvacrol, citral, and thymol appear effective against Prototheca species at the tested concentrations, and may thus be useful for environmental disinfection in barns.

The Development of a Multiplex PCR Assay for Fast and Cost-Effective Identification of the Five Most Significant Pathogenic Prototheca Species.

A multiplex PCR system (m-PCR) has been developed to accurately differentiate the five most important pathogenic Prototheca species, including the three species associated with infection in dairy cattle (P. ciferrii, P. blaschkeae, and P. bovis) and the two species associated with human infections (P. wickerhamii and P. cutis). The method is low-cost since it employs a simple "heat-shock" method in a TE buffer for DNA extraction. Furthermore, it requires only primers, a Taq polymerase, an agarose gel, and a molecular weight marker for identification. The method was based on published Prototheca cytochrome B sequences and was evaluated using reference strains from each of the five Prototheca species. The validity of the method was confirmed by identifying 50 strains isolated from milk samples. The specificity was tested in silico and with experimental PCR trials, showing no cross-reactions with other Prototheca species, as well as with bacteria, fungi, cows, algae, animals, or humans. The method could detect mixed infections involving two or three Prototheca species, providing a rapid test that delivers results within three hours.

Integration of transcriptomics, proteomics, and metabolomics data for the detection of the human pathogenic Prototheca wickerhamii from a One Health perspective.

Prototheca species are the only microalgae known to cause opportunistic infections in vertebrates and humans. Most cases of protothecosis in humans are caused by Prototheca wickerhamii, but knowledge of the pathogenicity and biology of Prototheca is limited. Globally, the diagnostic rate of Prototheca species infection is much lower than the actual rate of P. wickerhamii. The precise mechanisms underlying the pathogenesis of Prototheca infections remain unclear. In this study, we identified a strain of P. wickerhamii with atypical colony morphology. To reveal the morphological differences between P. wickerhamii S1 (mucous) and the molecular basis of its pathogenicity, the transcriptomics, proteomics, and metabolomics of two pathogenic P. wickerhamii strains and one environmental strain were analysed. Interestingly, mannan endo-1,4-β-mannosidase was significantly downregulated in P. wickerhamii S1, which contributes to a thinner cell wall in S1 compared to strains with typical colony morphology, and the toxicity of macrophages is reduced. Metabolite analysis revealed that the mucoid appearance of P. wickerhamii S1 may have been caused by an increase in linoleic acid, glycerol, and other metabolites. There is still a need to better understand the ecology, aetiology, and pathogenesis of P. wickerhamii, and in particular, its transmission between humans, animals, and the environment, from a One Health perspective.

Disseminated Protothecosis Due to Prototheca zopfii and Literature Review.

Prototheca species are achlorophyllic algae that are a rare cause of infection in humans. It most commonly causes localized cutaneous disease and rarely disseminated infection. Immunocompromised patients have the highest risk of disseminated protothecosis, with a higher mortality rate than localized cutaneous infections. At the species level, infections caused by Prototheca zopfii are reported less frequently than those caused by Prototheca wickerhamii. The diagnosis can be made using histopathology, culture, and molecular testing. There is no definitive evidence for an effective treatment, which currently consists of antifungals (primarily amphotericin B). With only a handful of cases of disseminated protothecosis reported worldwide that are caused by P. zopfii, we herein present an additional case of a postbone marrow transplant patient in the Midwest of the United States.

High Prevalence of Prototheca bovis Infection in Dairy Cattle with Chronic Mastitis in Ecuador.

The genus Prototheca, a unicellular, non-photosynthetic, yeast-like microalgae, is a pathogen of concern for the dairy industry. It causes bovine mastitis that currently cannot be cured, and hence generates significant economic losses in milk production. In this study, for the first time in Ecuador, we identify Prototheca bovis as the etiologic agent of chronic mastitis in dairy cattle. Milk samples (n = 458) of cows with chronic mastitis were cultured on Sabouraud Dextrose Agar (SDA). Microscopy and cytB gene sequencing were used to identify Prototheca, whereby Prototheca bovis was isolated from 15.1% (n = 69) of the milk samples, one of the highest infection rates that can be found in the literature in a "non-outbreak" situation. No other Prototheca species were found. We were unable to isolate the alga from environmental samples. We showed that P. bovis was relatively resistant to disinfectants used to sterilize milking equipment on the cattle farms where it was isolated. We discuss how to avoid future infection and also hypothesize that the real prevalence of Prototheca infection in bovine mastitis is probably much higher than what was detected. We recommend a protocol to increase the diagnostic yield in the bacteriology laboratory.

First Report of Molecular Identification of Prototheca zopfii Genotype 2 as Causative Agent of Bovine Mastitis in Navsari, South Gujarat, India

Prototheca species is emerged as an important causative agent for bovine mastitis worldwide, and is responsible for great economic losses for dairy industry. A 5-year-old HF cow was referred to the Veterinary Clinical Complex of the College, Navsari (India) with a complaint of watery milk with flakes, anorexia and drop in milk production. On physical examination, udder appeared warm and swollen, while milk consistency was thin, watery with flakes. Milk sample obtained aseptically from the cow was inoculated aerobically on Sabouraud dextrose agar and blood agar plates. Prototheca colonies grew in pure and luxuriant form from mastitic milk on both the plates after 48 h of incubation. Prototheca zopfii was identified on the basis of colony morphology, staining characters and biochemical reactions. Molecular confirmation of Prototheca zopfii genotype 2 was performed by employing polymerase chain reaction (PCR). This report presents first isolation and molecular identification of Prototheca zopfii genotype 2 from a case of bovine mastitis from Navsari region, South Gujarat, India.

Analysis of Prototheca and yeast species isolated from bulk tank milk collected in Tokachi District, Japan.

Bovine mastitis, a major infectious disease affecting milking cows, leads to reduced milk yield and quality, reduced animal welfare, and an increased need for culling. Although its major causative agents are bacteria, yeast species and achlorophyllous algae of the Prototheca genus are well known as causative agents of bovine refractory mastitis. Nevertheless, few studies have analyzed specific yeasts and Prototheca in this context. Herein, we present survey data of yeast species and Prototheca species isolated from bulk tank milk in the Tokachi district of Japan from April 2020 through March 2021. The species of 276 isolates were determined. Yeast species accounted for 184 isolates, of which Pichia kudriavzevii was the most prevalent species. Regarding Prototheca species, only Prototheca bovis was isolated (92 isolates). Prototheca bovis and Pichia kudriavzevii were detected throughout the year and were detected repeatedly on the same farm. Kluyveromyces marxianus was the second most frequently isolated yeast species after Pichia kudriavzevii. Candida parapsilosis, the fourth most frequently isolated yeast species, was found discontinuously. Analysis of monthly data indicated that Kluyveromyces marxianus and Candida parapsilosis were mainly found during the winter and summer months, respectively. Candida akabanensis and Pichia cactophila were the third and fifth most frequently isolated yeast species, respectively. They were detected repeatedly in bulk tank milk samples from the same farms. Results obtained from bulk tank milk underscore the prevalence of these species. These study results are expected to contribute to the elucidation of problematic yeast and Prototheca species.

Activity of biogenic silver nanoparticles against isolates of Prototheca species from bovine mastitis.

Prototheca spp. cause numerous infections in a wide variety of species, including treatment-unresponsive mastitis. Thus, the search for an effective therapy is essential. Silver nanoparticles are compounds with high therapeutic potential. This study aimed to evaluate the susceptibility profile and morphological changes in Prototheca spp. treated with biogenic silver nanoparticles (Bio-AgNP). The algaecide activity was evaluated in microplates by microdilution method, resulting in a MIC50 of 30 μg ml-1 and a MIC90 of 60 μg ml-1 . Scanning electron microscopy demonstrated changes in the surface of Prototheca bovis cells following treatment. The algaecide activity of Bio-AgNP suggests a therapeutic potential as a novel approach for the control of Prototheca spp. in bovine mastitis.

Protothecosis in the mucosa of the pharynx mimicking pharyngeal cancer in an immunocompetent individual: a case report

BackgroundProtothecosis is a rare infection in humans and animals caused by the achlorophyllic algae Prototheca species. More than half of the protothecosis cases are cutaneous infections, and most cases are observed in immunocompromised individuals.Case presentationWe report a case of Prototheca wickerhamii infection in the mucosa of the pharynx in a 53-year-old immunocompetent woman with an incidentally found mass lesion at the left tongue base. Histopathological findings of the mass lesion suggested cryptococcosis, but P. wickerhamii was identified from the oropharynx scrape culture based on DNA sequencing. After surgical resection, fosfluconazole treatment was initiated, and subsequently, treatment was switched to topical amphotericin B. The residual mass lesion did not deteriorate during the 4-month antifungal treatment and 1-year observational period.ConclusionsPrototheca species can be easily misdiagnosed as yeasts because of their morphological and pathological similarities. Prototheca, in addition to Cryptococcus should be considered if slow-growing, large Gram-positive organisms are encountered. Lactophenol cotton blue staining of the colony helps distinguish these organisms. Further study is needed to determine the appropriate treatment according to the infection focus.

Prototheca-ID: a web-based application for molecular identification of Prototheca species.

The genus Prototheca houses unicellular, achlorophyllous, yeast-like algae, widely distributed in the environment. Protothecae are the only known plants that have repeatedly been reported to infect vertebrates, including humans. Although rare, protothecosis can be clinically demanding, with an unpredictable and treatment-resistant behavior. Accurate identification of Prototheca species relies upon DNA sequence-based typing of the mitochondrially encoded CYTB gene. However, no bioinformatic tool for the processing and analyzing of protothecal sequence data exists. Moreover, currently available sequence databases suffer from a limited number of records and lack of or flawed sequence annotations, making Prototheca identification challenging and often inconclusive. This report introduces the Prototheca-ID, a user-friendly, web-based application providing fast and reliable speciation of Prototheca isolates. In addition, the application offers the users the possibility of depositing their sequences and associated metadata in a fully open Prototheca-ID database, developed to enhance research integrity and quality in the field of Protothecae and protothecosis. Database URL: The Prototheca-ID application is available at https://prototheca-id.org

Prototheca bovis, a unicellular achlorophyllous trebouxiophyte green alga in the healthy human intestine.

Introduction. Prototheca species are non-photosynthetic trebouxiophyte algae ubiquitously distributed in nature and can be found in sewage and soil. This microbial eukaryote causes human protothecosis in immunocompromised individuals. Thus, Prototheca presence in the stool of individuals without gastrointestinal symptoms has been reported only rarely.Hypothesis/Gap statement. There is an absence of detailed characterization of human Prototheca isolates.Aim. The aim of this study was to perform morphological and molecular characterization of Prototheca isolates obtained from human stool.Methodology. Prototheca was isolated from faecal samples of four individuals living in a rural area in Thailand. A combination of bioimaging along with molecular and bioinformatics tools was used to characterize the four strains. The growth rate was tested using four media and three temperature conditions. Phylogenetic analysis using the small subunit ribosomal RNA (SSU rRNA) and cytochrome b (cytb) was also performed.Results. Static and live microscopy demonstrated the various life stages of Prototheca and its major defining cellular characteristics. An optimized DNA extraction methodology that improves DNA yield is provided. Partial fragments of the SSU rRNA and cytb genes were obtained. Phylogenetic analysis placed all four strains in the clade with Prototheca bovis. More broadly, Prototheca was not monophyletic but split into at least two distinct clades instead.Conclusion. The results represent the first molecular characterization of Prototheca in Thailand. The study provides insight into transmission dynamics of the organism and potential caveats in estimating the global prevalence of Prototheca. These will spearhead further investigations on Prototheca occurrence in rural areas of both industrialized and developing nations.

Prevalence of Prototheca and fungal contamination of bulk tank milk of industrial dairy cattle herds in Iran

Background: Prototheca Spp. belongs to family Chlorellaceae and can cause mastitis in dairy cattle herds. Several Prototheca species could be isolated in different parts of the dairy herds. Objectives: The objectives were to determine: (1) the investigation status of bulk tank milk Prototheca Spp prevalence in industrial dairy cattle herds in different area of Iran. And (2). To evaluate the fungal agent prevalence of bulk tank milk of dairy cattle herds in different seasons. Materials and Methods: Bulk tank milk for the presence of Prototheca and fungal agents from 165 industrial dairy cattle herds complexes were tested. The population of each herd were about 850–3500 Holstein dairy cows. The samples were taken from 32 cities and 16 provinces of Iran, respectively. In total, almost 66800 Holstein dairy cows represented. The population of lactating cows included about 25800. Samples were cultured on sabouraud glucose agar (SGA) medium with chloramphenicol (100 mg / l). Results: Only 8 (4.84%) out of 165 samples were isolated for Prototheca Spp. It had been isolated in different seasons. They were 2, 4, 1 and 1 samples in spring, summer, autumn and winter, respectively. Candida, Fusarium and Trichosporon Spp. were isolated from 137, 2 and 1 samples, respectively. According to the obtained results Prototheca and fungal agents are present in bulk tank milk of industrial dairy cattle herds in different provinces of Iran. Conclusion: It is concluded that exposure to Prototheca and different fungal agents could be common in the dairy cattle herds in Iran. The results are important as dairy cattle health and human sanitation hazards.

MALDI-TOF MS identification of Prototheca algae associated with bovine mastitis.

We evaluated the use of MALDI-TOF MS for the identification of 3 major, dairy-associated Prototheca species, namely, Prototheca bovis (formerly P. zopfii genotype 2), P. blaschkeae, and P. ciferrii (formerly P. zopfii genotype 1). The MALDI-TOF MS spectra established for those species were introduced into the reference spectra library of the Bruker Biotyper MALDI-TOF MS analysis software. Next, 31 Prototheca isolates from Holstein cows with mastitis, from herds located in the midwestern area of São Paulo State, Brazil, were subjected to MALDI-TOF MS profiling. MALDI-TOF MS allowed identification of 22 of 27 P. bovis and 3 of 4 P. blaschkeae isolates with scores >2.0, with 5 of 27 P. bovis and 1 of 4 P. blaschkeae isolates identified only to the genus level. With our extended algae database, MALDI-TOF MS can contribute to quick and effective speciation of Prototheca from mastitis cases.

Prototheca spp. co-infections and their virulence factors in human protothecosis in Hillah city, Iraq

BackgroundThe objective of the present study was to investigate coinfection of Pseudomonas aeruginosa and Prototheca species and other microorganisms in human protothecosis infections. MethodsThe current study included a collection of 275 clinical samples in Hillah, Iraq. Following bacterial identification, virulence factors genes were screened using polymerase chain reaction (PCR) technique. ResultsThe results showed four types of Prototheca species Prototheca zopfii genotype1, Prototheca zopfii genotype2, Prototheca stagnora and Prototheca blaschkeae responsible for human protothecosis infections in burns, urinary tract infection (UTI), leukemia and dialysis. Furthermore, this study clarified coinfection of Prototheca spp. and P. aeruginosa. The predominant Prototheca spp. virulence genes included lasR (41.7%), ecfXECF (26.6%), rhlR (19.6%) and toxAETA (21.3%), with significant difference (P-0.047). furthermore, there was an association between Prototheca spp. and P. aeruginosa virulence genes such as PA-GS 16sRNA, lasB ELA520, toxAETA397, phzM PYO330, ecfXECF164, lasR, rhlR, PlcH. ConclusionThis study revealed the existence of Prototheca spp. carrying various virulence genes among burns and UTI in Hillah city, Iraq. Bacterial co-infections exacerbate the severity of infections, particularly those potential nosocomial pathogens. Identification of P. aeruginosa virulence genes among Prototheca spp. highlighted their role in severe infections.

Molecular characterization of Prototheca in 11 symptomatic dogs.

Protothecosis is an uncommon disease caused by algae of the genus Prototheca. In dogs, the infection is usually first localized to the colon but has the propensity to later disseminate hematogenously to many other organs, with marked tropism for the eyes and central nervous system. Diagnosis is established by culture and/or evidence of Prototheca organisms in cytologic or histologic preparations. Species characterization, however, requires molecular investigations. Our laboratory set up a real-time PCR targeting portion D1/D2 of the 28S rRNA for identification of Prototheca species from both positive cultures (of rectal swabs and urine) and formalin-fixed, paraffin-embedded tissue. Prototheca bovis, P. ciferrii, and P. wickerhamii were characterized in 11 dogs with systemic or cutaneous protothecosis. Prototheca identifications were phylogenetically consistent with the new taxonomy proposed for this genus based on the mitochondrial cytochrome b gene. As a pilot study, we screened feces and rectal scrapes from 200 asymptomatic dogs, using 2 cohorts of stray and owned animals, to determine the prevalence of intestinal carriage of Prototheca spp. The Prototheca-negative results from both cohorts of healthy dogs suggest that predisposing factors related to the host probably contribute more to the acquisition of clinical disease than exposure to contaminated environments.

Sequencing and Analysis of the Complete Organellar Genomes of Prototheca wickerhamii.

Of the Prototheca genus, Prototheca wickerhamii has the highest clinical significance in humans. However, neither nuclear nor organellar genomes of this species were sequenced until now. The hitherto determined and analyzed mitochondrial and plastid genomes of the alleged P. wickerhamii species belong in fact to another species, recently named Prototheca xanthoriae. This study provides a first insight into the organellar genomes of a true P. wickerhamii (type strain ATCC 16529). The P. wickerhamii mitochondrion had a 53.8-kb genome, which was considerably larger than that of Prototheca ciferrii (formerly Prototheca zopfii gen. 1) and Prototheca bovis (formerly Prototheca zopfii gen. 2), yet similarly functional, with the differences in size attributable to a higher number of introns and the presence of extra unique putative genes. The 48-kb plastid genome of P. wickerhamii, compared to autotrophic Trebouxiophyceae, was highly reduced due to the elimination of the photosynthesis-related genes. The gene content of the plastid genome of P. wickerhamii was, however, very similar to other colorless Prototheca species. Plastid genome-based phylogeny reinforced the polyphyly of the genus Prototheca, with Helicosporidium and Auxenochlorella branching within clades of Prototheca species. Phylogenetic reconstruction also confirmed the close relationship of P. wickerhamii and P. xanthoriae, which is reflected in the synteny of their organellar genomes. Interestingly, the entire set of atp genes was lost in P. wickerhamii plastid genome while being preserved in P. xanthoriae.

In vitro algicidal effect of polypyrrole on Prototheca species isolates from bovine mastitisAlgicidal activity of polypyrrole on Prototheca spp.

Algae of the genus Prototheca are microorganisms involved in the occurrence of diseases in humans and animals. In bovine species, Prototheca spp. cause environmental mastitis, productive losses in dairy herds, mainly leading to the discard of infected cows. Currently, there are no effective anti-Prototheca spp. drugs to combat this infection. Thus, the search for an efficacious therapy for Prototheca spp. infections have become essential. Highly soluble polypyrrole (Ppy) is a molecule with known antimicrobial activity. This study aimed to characterize Prototheca spp. isolates from bovine mastitis as well as to evaluate the susceptibility profile and to verify the morphological alterations on Prototheca spp. isolates treated with Ppy. In this research, 36 Brazilian isolates of Prototheca spp. were characterized by restriction fragment length polymorphism polymerase chain reaction (RFLP-PCR) assay for the mitochondrial cytB gene. Additionally, Ppy algicidal activity against these isolates of Prototheca spp. was assessed by minimal microbicidal concentration method in microplates. Further, scanning electron microscopy (SEM) was performed in order to verify the morphological alterations on Prototheca spp. isolates in response to Ppy. The isolates were characterized as belonging to Prototheca zopfii genotype 2 (35/36) and Prototheca blaschkeae (1/36). Ppy had an algicidal effect on all isolates tested at concentrations ranging from 15.625μg ml-1 to 62.5μg ml-1. SEM showed changes on planktonic and sessile P. zopfii, including a decrease of the number of cells with the presence of an amorphous substance involving the cells. The algicidal activity of Ppy suggests the therapeutic potential of this molecule in the prevention and treatment of Prototheca spp. in bovine mastitis.

In vitro algaecid effect of itraconazole and ravuconazole on Prototheca species.

Protothecosis is a rare infection in humans, dogs, and cats, and its causative agent is Prototheca species, which consists of achlorophyllic algae that are ubiquitous in natural environments and hosts intestinal flora. Ravuconazole (RVZ) is a new available human azole drug in Japan since 2018 and broad-spectrum antifungal agent. In the present study, the in vitro susceptibility of clinical and environmental isolates of P. wickerhamii, P. zopfii, and P. blaschkeae to itraconazole (ITZ) voriconazole (VRZ), posaconazole (PCZ), and RVZ. RVZ was more potent than the other azoles against Protheca species and has considerable potential for use as a therapeutic agent for human and animal protothecosis.

Algicidal effect of blue light on pathogenic Prototheca species.

Prototheca spp. are pathogenic algae with important zoonotic potential. Most importantly, these algae often infect dairy cattle. Since there is no effective therapy against the algae, the standard recommendation is the disposal or culling of infected cows to avoid outbreaks. This study investigated the ability of blue light to inactivate pathogenic Prototheca species. Blue LED light (λ = 410 nm) was used to inactivate in vitro suspensions of P. zopfii genotypes 1 and 2, and P. blaschkeae. Our results showed that blue light irradiation induced a strain-specific dose-dependent algicidal effect against all tested strains. P. zopfii genotype 1, was more sensitive than genotype 2 and P. blaschkeae was the most tolerant. Even though we observed different inactivation kinetics, all strains presented significant photoinactivation levels within feasible procedure periods. Therefore, we conclude that blue light irradiation offers promising potential for the development of novel technologies that control contaminations and infections caused by Prototheca spp.