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Protothecosis is a rare but emerging infectious disease, caused by algae from the genus Prototheca. It presents predominantly as cutaneous lesions and poses a diagnostic challenge owing to its diverse clinical presentation. Typically, it occurs in exposed areas of the skin, associated with trauma. Diagnosis relies mainly on histopathology and culture. This article presents an elderly diabetic woman with cutaneous protothecosis from Rio de Janeiro, Brazil. We emphasize clinical features, histopathology, and successful treatment with itraconazole, along with a brief review of the 12 previously reported cases from Brazil.

Canine protothecosis is a rare disease caused by saprophytic unicellular achlorophyllous aerobic algae that are ubiquitous in the environment. We report a novel case of neurological and cardiological manifestations associated with disseminated protothecosis. An adult spayed female Boxer dog was presented with a 1‐week history of anorexia, progressive central vestibular signs, and a Grade III/VI systolic heart murmur. Magnetic resonance (MR) imaging revealed obstructive hydrocephalus at the level of the mesencephalic aqueduct, while echocardiography and elevated troponin levels suggested an infiltrative cardiomyopathy. No obvious cause was identified. Cerebrospinal fluid (CSF) collection was not performed due to associated procedural risks. Despite receiving symptomatic treatment and maintaining stability for 3 weeks, the dog eventually suffered cardiorespiratory arrest. Postmortem examination revealed disseminated protothecosis, predominantly affecting the heart and brain. We recommend that in cases where the cause of obstructive hydrocephalus is unclear, especially when CSF collection is not feasible, a comprehensive diagnostic method should be implemented. This includes meticulous investigations to identify infected tissues, followed by sampling and performing cytology/histology and culture tests to confirm the presence of the algal organism. Early diagnosis may allow early treatment, although long‐term prognosis remains largely unfavorable due to the absence of effective treatments. [ABSTRACT FROM AUTHOR]

Prototheca wickerhamii is a rare cause of cutaneous and systemic infection that requires long treatment courses with potentially toxic medications. We describe a patient with cutaneous protothecosis refractory to triazole monotherapy who experienced clinical and radiographic improvement with the novel oral lipid nanocrystal formulation of amphotericin B without experiencing toxicity. [ABSTRACT FROM AUTHOR]

The aim of this study was to determine the prevalence of Streptococcus (Sc.) agalactiae, Prototheca spp., Staphylococcus (S.) aureus, and especially methicillin-resistant S. aureus as well as Myco-plasmopsis (M.) spp. and M. bovis in bulk tank milk (BTM) on dairy farms in Lower Saxony, Germany. BTM samples were collected in January 2023 from 208 selected dairy farms. The samples were quantitatively culturally analyzed for S. aureus and Prototheca spp. Presumptive S. aureus colonies were further confirmed by MALDI-TOF. Presumptive Prototheca spp. colonies were confirmed by light microscopy. Sc. agalactiae and Mycoplasmopsis spp. were detected by real-time polymerase chain reaction (rtPCR). Sc. agalactiae was detected in two herds (1% (Confidence Interval 95% (CI) 0.3–3.4)). S. aureus was confirmed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) in 38 herds (18.3% (CI 13.6–24.1)), assuming a threshold of >10 cfu/mL milk. A total of 154 isolates identified as S. aureus by MALDI-TOF were transferred to agar with added oxacillin for resistance testing, of which 19 isolates (12.3% (CI 8–18.5)) showed growth. The 19 isolates came from eight different farms (3.8% (2–7.4)). Prototheca spp. were identified in 13 herds (6.3% (CI 3.7–10.4)). Mycoplasmopsis spp. were detected by PCR in 18 herds (8.7% (CI 5.5–13.3)). Of these, M. bovis was present in three herds (1.4% (0.5–4.2)). The herd prevalence of Sc. agalactiae in BTM appears to be at low levels in the sampled area. The prevalence of Mycoplasmopsis spp. in the herds was higher than expected compared to previous studies. It is interesting to note that the percentage of M. bovis in the total Mycoplasmopsis spp. was only 16.7%. [ABSTRACT FROM AUTHOR]

Background: Protothecosis is an infection of humans and animals caused by a rare conditionally pathogenic fungus (prototheca). It can occur in immunocompromised or normal patients. Aims: To describe the epidemiology of prototheca infection in China. Methods: We report a case of successful treatment of cutaneous protothecosis with fluconazole and analyzed the epidemiological characteristics, risk factors, clinical manifestations, diagnosis, treatment and prognosis of prototheca infections in China. Results: We describe this case and 29 cases of prototheca infections in China. At present, Prototheca wickerhamii (Pw) infection is the most common infection in China, and single or combined itraconazole is the preferred treatment. Conclusions: These results provide detailed information and relevant clinical treatment strategies for the diagnosis and treatment of protothecosis in China. [ABSTRACT FROM AUTHOR]

ABSTRACT: Anatomopathological descriptions of infections caused by saprophytic algae in dogs are rare. This study reported the anatomopathological and microbiological findings suggestive of disseminated protothecosis in a dog residing in the state of Espírito Santo, Brazil. During necropsy, macroscopic alterations were observed in peripheral lymph nodes, heart, kidneys, pancreas, and lungs. Microbiological, cytological, and histopathological examinations were performed. Cytology and histopathology revealed piogranulomatous inflammation associated with sporangia and endospores, while microbiological culture suggested the growth of Prototheca spp.. These anatomopathological and microbiological findings were crucial for diagnosing and documenting the first case of disseminated Prototheca spp. compatible infection in a dog in the state of Espírito Santo, Brazil.

ABSTRACT The majority of the nearly 10,000 described species of green algae are photoautotrophs; however, some species have lost their ability to photosynthesize and become obligate heterotrophs that rely on parasitism for survival. Two high-quality genomes of the heterotrophic algae Prototheca zopfii Pz20 and Pz23 were obtained using short- and long-read genomic as well as transcriptomic data. The genome sizes were 31.2 Mb and 31.3 Mb, respectively, and contig N50 values of 1.99 Mb and 1.26 Mb. Although P. zopfii maintained its plastid genome, the transition to heterotrophy led to a reduction in both plastid and nuclear genome size, including the loss of photosynthesis-related genes from both the nuclear and plastid genomes and the elimination of genes encoding for carotenoid oxygenase and pheophorbide an oxygenase. The loss of genes, including basic leucine-zipper (bZIP) transcription factors, flavin adenine dinucleotide-linked oxidase, and helicase, could have played a role in the transmission of autotrophy to heterotrophs and in the processes of abiotic stress resistance and pathogenicity. A total of 66 (1.37%) and 73 (1.49%) genes were identified as potential horizontal gene transfer events in the two P. zopfii genomes, respectively. Genes for malate synthase and isocitrate lyase, which are horizontally transferred from bacteria, may play a pivotal role in carbon and nitrogen metabolism as well as the pathogenicity of Prototheca and non-photosynthetic organisms. The two high-quality P. zopfii genomes provide new insights into their evolution as obligate heterotrophs and pathogenicity.IMPORTANCEThe genus Prototheca, characterized by its heterotrophic nature and pathogenicity, serves as an exemplary model for investigating pathobiology. The limited understanding of the protothecosis infectious disease is attributed to the lack of genomic resources. Using HiFi long-read sequencing, both nuclear and plastid genomes were generated for two strains of P. zopfii. The findings revealed a concurrent reduction in both plastid and nuclear genome size, accompanied by the loss of genes associated with photosynthesis, carotenoid oxygenase, basic leucine-zipper (bZIP) transcription factors, and others. The analysis of horizontal gene transfer revealed the presence of 1.37% and 1.49% bacterial genes, including malate synthase and isocitrate lyase, which play crucial roles in carbon and nitrogen metabolism, as well as pathogenicity and obligate heterotrophy. The two high-quality P. zopfii genomes represent valuable resources for investigating their adaptation and evolution as obligate heterotrophs, as well as for developing future prevention and treatment strategies against protothecosis.

The aim of this study was to determine the prevalence of Streptococcus (Sc.) agalactiae, Prototheca spp., Staphylococcus (S.) aureus, and especially methicillin-resistant S. aureus as well as Myco-plasmopsis (M.) spp. and M. bovis in bulk tank milk (BTM) on dairy farms in Lower Saxony, Germany. BTM samples were collected in January 2023 from 208 selected dairy farms. The samples were quantitatively culturally analyzed for S. aureus and Prototheca spp. Presumptive S. aureus colonies were further confirmed by MALDI-TOF. Presumptive Prototheca spp. colonies were confirmed by light microscopy. Sc. agalactiae and Mycoplasmopsis spp. were detected by real-time polymerase chain reaction (rtPCR). Sc. agalactiae was detected in two herds (1% (Confidence Interval 95% (CI) 0.3–3.4)). S. aureus was confirmed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) in 38 herds (18.3% (CI 13.6–24.1)), assuming a threshold of >10 cfu/mL milk. A total of 154 isolates identified as S. aureus by MALDI-TOF were transferred to agar with added oxacillin for resistance testing, of which 19 isolates (12.3% (CI 8–18.5)) showed growth. The 19 isolates came from eight different farms (3.8% (2–7.4)). Prototheca spp. were identified in 13 herds (6.3% (CI 3.7–10.4)). Mycoplasmopsis spp. were detected by PCR in 18 herds (8.7% (CI 5.5–13.3)). Of these, M. bovis was present in three herds (1.4% (0.5–4.2)). The herd prevalence of Sc. agalactiae in BTM appears to be at low levels in the sampled area. The prevalence of Mycoplasmopsis spp. in the herds was higher than expected compared to previous studies. It is interesting to note that the percentage of M. bovis in the total Mycoplasmopsis spp. was only 16.7%.

ABSTRACT: Better understanding of the molecular mechanisms behind bovine mastitis is fundamental for improving the management of this disease, which continues to be of major concern for the dairy industry, especially in its subclinical form. Disease severity and progression depend on numerous aspects, such as livestock genetics, and the interaction between the causative agent, the host, and the environment. In this context, epigenetic mechanisms have proven to have a role in controlling the response of the animal to inflammation. Therefore, in this study we aimed to explore genome-wide DNA methylation of milk somatic cells (SC) in healthy cows (n = 15) and cows affected by naturally occurring subclinical mastitis by Streptococcus agalactiae (n = 12) and Prototheca spp. (n = 11), to better understand the role of SC methylome in the host response to disease. Differentially methylated regions (DMR) were evaluated comparing: (1) Strep. agalactiae-infected versus healthy; (2) Prototheca-infected versus healthy, and (3) mastitis versus healthy and (4) Strep. agalactiae-infected versus Prototheca-infected. The functional analysis was performed at 2 levels. To begin with, we extracted differentially methylated genes (DMG) from promoter DMR, which were analyzed using the Cytoscape ClueGO plug-in. Coupled with this DMG-driven approach, all the genes associated with promoter-methylated regions were fed to the Pathifier algorithm. From the DMR analysis, we identified 1,081 hypermethylated and 361 hypomethylated promoter regions in Strep. agalactiae-infected animals, while 1,514 hypermethylated and 358 hypomethylated promoter regions were identified in Prototheca-infected animals, when compared with the healthy controls. When considering infected animals as a whole group (regardless of the pathogen), we found 1,576 hypermethylated and 460 hypomethylated promoter regions. Both pathogens were associated with methylation differences in genes involved in pathways related to meiosis, reproduction and tissue remodeling. Exploring the whole methylome, in subclinically infected cows we observed a strong deregulation of immune-related pathways, such as nuclear factor kB and toll-like receptors signaling pathways, and of energy-related pathways such as the tricarboxylic acid cycle and unsaturated fatty acid biosynthesis. In conclusion, no evident pathogen-specific SC methylome signature was detected in the present study. Overall, we observed a clear regulation of host immune response driven by DNA methylation upon subclinical mastitis. Further studies on a larger cohort of animals are needed to validate our results and to possibly identify a unique SC methylome that signifies pathogen-specific alterations.

Better understanding of the molecular mechanisms behind bovine mastitis is fundamental for improving the management of this disease, which continues to be of major concern for the dairy industry, especially in its subclinical form. Disease severity and progression depend on numerous aspects, such as livestock genetics, and the interaction between the causative agent, the host, and the environment. In this context, epigenetic mechanisms have proven to have a role in controlling the response of the animal to inflammation. Therefore, in this study we aimed to explore genome-wide DNA methylation of milk somatic cells (SC) in healthy cows (n = 15) and cows affected by naturally occurring subclinical mastitis by Streptococcus agalactiae (n = 12) and Prototheca spp. (n = 11), to better understand the role of SC methylome in the host response to disease. Differentially methylated regions (DMR) were evaluated comparing: (1) Strep. agalactiae -infected versus healthy; (2) Prototheca -infected versus healthy, and (3) mastitis versus healthy and (4) Strep. agalactiae -infected versus Prototheca -infected. The functional analysis was performed at 2 levels. To begin with, we extracted differentially methylated genes (DMG) from promoter DMR, which were analyzed using the Cytoscape ClueGO plug-in. Coupled with this DMG-driven approach, all the genes associated with promoter-methylated regions were fed to the Pathifier algorithm. From the DMR analysis, we identified 1,081 hypermethylated and 361 hypomethylated promoter regions in Strep. agalactiae -infected animals, while 1,514 hypermethylated and 358 hypomethylated promoter regions were identified in Prototheca -infected animals, when compared with the healthy controls. When considering infected animals as a whole group (regardless of the pathogen), we found 1,576 hypermethylated and 460 hypomethylated promoter regions. Both pathogens were associated with methylation differences in genes involved in pathways related to meiosis, reproduction and tissue remodeling. Exploring the whole methylome, in subclinically infected cows we observed a strong deregulation of immune-related pathways, such as nuclear factor kB and toll-like receptors signaling pathways, and of energy-related pathways such as the tricarboxylic acid cycle and unsaturated fatty acid biosynthesis. In conclusion, no evident pathogen-specific SC methylome signature was detected in the present study. Overall, we observed a clear regulation of host immune response driven by DNA methylation upon subclinical mastitis. Further studies on a larger cohort of animals are needed to validate our results and to possibly identify a unique SC methylome that signifies pathogen-specific alterations. [ABSTRACT FROM AUTHOR]

Yeast infections are challenging human and animal medicine due to low rates of detection and the emergence of unknown ecology isolates. The aim of this study was to verify the biochemical identification of yeasts and yeast-like microorganisms obtained from animals comparing the results with chromogenic media and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF MS). Between January and August 2023, yeast and yeast-like isolates from samples of animals with suspicion of mycosis were identified using Vitek® 2 Compact, Brilliance® Candida Agar and MALDI Biotyper® MSP. A total of 39 cases were included, and 45 isolations were obtained. Cryptococcus neoformans (15.5%, 7/45), Meyerozyma guilliermondii (13.3%, 6/45), Candida parapsilosis (11.1%, 5/45), Candida albicans and Candida tropicalis (8.9%, each one 4/45) were the most identified organisms. There was full agreement with the three identification methods in 71.1% (32/45) of the isolates, disagreement on species in 17.8% (8/45), disagreement on genus and species in 6.7% (3/45) and, in 4.4% (2/45), there was no matched pattern in MALDI-TOF to compare the results. Biochemical methods are a good option in laboratories where proteomics are not available, and chromogenic media enhances diagnostics by detecting mixed infections. Surveillance must be implemented to improve the detection of agents shared between humans and animals. [ABSTRACT FROM AUTHOR]

Simple Summary: The genus Prototheca encompasses unicellular algae that are achlorophyllous and widespread in the environment. The genus is now included in the family Chlorellaceae, belonging to the order Chlorellales, which is included in the class, Trebouxiophyceae. Prototheca have repeatedly been reported to infect vertebrates. Cattle, dogs, and cats are the unique domestic animals in which Prototheca spp. have been reported, despite sporadic detection in goats, horses, and non-domesticated animals. Prototheca spp. have been reported to colonize different districts of the human body. Cats with protothecosis usually display a cutaneous disease, whereas dogs may develop both cutaneous and systemic forms. In this report, we identified molecularly Prototheca spp. in a cat with neurological signs. The animal presented a suspected diagnosis of multifocal lymphoma, and eventual immunological disorders/suppression likely triggered systemic diffusion of the achlorophyllic alga. Despite protothecosis not being regarded as a zoonosis, algal infections of animals should be recognized as indicators or sentinels of environmental risks for humans. Prototheca infections are rare in cats, and they are usually associated with cutaneous or subcutaneous infections by P. wickerhamii, with no evidence of neurological signs or systemic disease. In this study, we report the identification of prototheca in the cerebrospinal fluid (CSF) of a cat with neurological symptoms. Fourteen CSF samples were gathered from cats presented with neurological disease between 2012 and 2014. The inclusion criteria for the samples were an increase in CSF protein and cell number (pleocytosis), suggestive of an infectious inflammatory status of the central nervous system (CNS). Nine samples fulfilled the inclusion criteria (inflammatory samples), while five samples, used as control, did not (non-inflammatory samples). All the samples were screened molecularly for different pathogens associated with CNS disease in cats, including prototheca. Out of 14 CSF samples, only one inflammatory sample tested positive for prototheca. Upon sequence and phylogenetic analysis of the amplicon, the strain was characterized as P. bovis. This report is the first documented evidence of prototheca in the cerebrospinal fluid of a cat with neurological signs. Prototheca should be considered in the diagnostics procedures on the CNS of cats presented with infectious diseases. [ABSTRACT FROM AUTHOR]

Protothecosis and chlorellosis are sporadic algal diseases that can affect small ruminants. In goats, protothecosis is primarily associated with lesions in the nose and should be included in the differential diagnosis of causes of rhinitis. In sheep, chlorellosis causes typical green granulomatous lesions in various organs. Outbreaks of chlorellosis have been reported in sheep consuming stagnant water, grass from sewage-contaminated areas, and pastures watered by irrigation canals or by effluents from poultry-processing plants. Prototheca and Chlorella are widespread in the environment, and environmental and climatic changes promoted by anthropogenic activities may have increased the frequency of diseases produced by them. The diagnosis of these diseases must be based on gross, microscopic, and ultrastructural lesions, coupled with detection of the agent by immunohistochemical-, molecular-, and/or culture-based methods.

A case study describes a 6-year-old mixed-breed dog presenting with sudden blindness, mydriasis, uveitis and bilateral conjunctival hyperemia. Examination revealed white subretinal transudate mimicking granulomas, along with concurrent intermittent hematochezia and pain on abdominal palpation, suggestive of protothecosis. Cytology revealed unicellular organisms consistent with Prototheca species, confirming the diagnosis.

Abstract Background The Prototheca algae have recently emerged as an important cause of bovine mastitis globally. Isolates from bovine mastitis in several countries were nearly all identified as P. bovis, suggesting that it was the main causative agent of bovine protothecal mastitis. The aim of the present study was to evaluate the presence and isolation of Prototheca spp. in dairy farms, detect the genetic diversity among strains, determine the capacity of producing biofilm and their resistance to antifungal and antimicrobial drugs. Results A total of 48 Prototheca isolates from four different farms were randomly selected to be investigated. Multiplex PCR showed all isolated colonies were Prototheca bovis. Performing RAPD-PCR by using OPA-4 primer, it was revealed that there was a clear amplification pattern. Different levels of biofilm production were observed among strains. Among 48 isolates, only 4 of them (8.33%) showed strong biofilm production. By using E-test strips, amphotericin B was able to inhibit the growth of all the strains tested. Disc diffusion method used for antimicrobial sensitivity test showed that the highest activity was demonstrated by gentamicin and colistin with 95.83% (46/48) and 89.58% (43/48) of sensitive strains, respectively. Conclusions The present study showed that RAPD-PCR was a rapid tool for discriminating P. bovis strains. Also, gentamicin and colistin can be considered as potential antimicrobial drugs which can prevent the growth of the mentioned strains in vitro, although there is no effective clinical treatment yet. Further studies are needed in order to detect an effective clinical therapy considering biofilm production by Prototheca spp. and their probable role in Prototheca pathogenicity.

ABSTRACT: 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.

Prototheca is a microalgae known to cause infections in humans, with protothecosis most commonly presenting as olecranon bursitis or localized soft tissue infection. Disseminated disease can be seen in immunocompromised patients. In this retrospective single-institution case series, we describe our experience with 7 patients with Prototheca infections. [ABSTRACT FROM AUTHOR]

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-b-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. [ABSTRACT FROM AUTHOR]

Yeast infections are challenging human and animal medicine due to low rates of detection and the emergence of unknown ecology isolates. The aim of this study was to verify the biochemical identification of yeasts and yeast-like microorganisms obtained from animals comparing the results with chromogenic media and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF MS). Between January and August 2023, yeast and yeast-like isolates from samples of animals with suspicion of mycosis were identified using Vitek® 2 Compact, Brilliance® Candida Agar and MALDI Biotyper® MSP. A total of 39 cases were included, and 45 isolations were obtained. Cryptococcus neoformans (15.5%, 7/45), Meyerozyma guilliermondii (13.3%, 6/45), Candida parapsilosis (11.1%, 5/45), Candida albicans and Candida tropicalis (8.9%, each one 4/45) were the most identified organisms. There was full agreement with the three identification methods in 71.1% (32/45) of the isolates, disagreement on species in 17.8% (8/45), disagreement on genus and species in 6.7% (3/45) and, in 4.4% (2/45), there was no matched pattern in MALDI-TOF to compare the results. Biochemical methods are a good option in laboratories where proteomics are not available, and chromogenic media enhances diagnostics by detecting mixed infections. Surveillance must be implemented to improve the detection of agents shared between humans and animals.

ABSTRACT: In this study, we investigated associations among subclinical intra-mammary infection (IMI) and quarter-level milk composition, udder health indicators, and cheesemaking traits. The dataset included records from 450 Holstein cows belonging to three dairy herds. After an initial screening (T0) to identify animals infected by Streptococcus agalactiae, Streptococcus uberis, Staphylococcus aureus, and Prototheca spp., 613 quarter milk samples for 2 different sampling times (T1 and T2, 1 mo after T1) were used for analysis. Milk traits were analyzed using a hierarchical linear mixed model including the effects of days in milk, parity and herd, and bacteriological and inflammatory category [culture negative with somatic cell count (SCC)

Prototheca infections are rare in cats, and they are usually associated with cutaneous or subcutaneous infections by P. wickerhamii, with no evidence of neurological signs or systemic disease. In this study, we report the identification of prototheca in the cerebrospinal fluid (CSF) of a cat with neurological symptoms. Fourteen CSF samples were gathered from cats presented with neurological disease between 2012 and 2014. The inclusion criteria for the samples were an increase in CSF protein and cell number (pleocytosis), suggestive of an infectious inflammatory status of the central nervous system (CNS). Nine samples fulfilled the inclusion criteria (inflammatory samples), while five samples, used as control, did not (non-inflammatory samples). All the samples were screened molecularly for different pathogens associated with CNS disease in cats, including prototheca. Out of 14 CSF samples, only one inflammatory sample tested positive for prototheca. Upon sequence and phylogenetic analysis of the amplicon, the strain was characterized as P. bovis. This report is the first documented evidence of prototheca in the cerebrospinal fluid of a cat with neurological signs. Prototheca should be considered in the diagnostics procedures on the CNS of cats presented with infectious diseases.

La prototecosis es una enfermedad causada por algas saprófitas, no clorofílicas, unicelulares y oportunistas del género Prototheca, que afecta, principalmente, a animales con inmunodeficiencias. En caninos, con la forma intestinal, se puede presentar inicialmente como diarrea sanguinolenta, pudiendo progresar a un cuadro sistémico; mientras que en felinos, generalmente, genera lesiones cutáneas. En Costa Rica, P. zopfii es la especie que se ha identificado con mayor frecuencia, y P. wickerhamii, una vez. El diagnóstico de esta enfermedad se puede realizar mediante distintas técnicas como: citología, histopatología, endoscopía, cultivo, reacción en cadena de polimerasa (PCR), método bioquímico, entre otros. El tratamiento recomendado es anfotericina B e itraconazol, los cuales son efectivos, principalmente, en felinos. Sin embargo, en caninos, con presentación sistémica, no existe un tratamiento efectivo. En caso de lesiones cutáneas, se recomienda la resección quirúrgica.

Background: The Prototheca algae have recently emerged as an important cause of bovine mastitis globally. Isolates from bovine mastitis in several countries were nearly all identified as P. bovis, suggesting that it was the main causative agent of bovine protothecal mastitis. The aim of the present study was to evaluate the presence and isolation of Prototheca spp. in dairy farms, detect the genetic diversity among strains, determine the capacity of producing biofilm and their resistance to antifungal and antimicrobial drugs. Results: A total of 48 Prototheca isolates from four different farms were randomly selected to be investigated. Multiplex PCR showed all isolated colonies were Prototheca bovis. Performing RAPD-PCR by using OPA-4 primer, it was revealed that there was a clear amplification pattern. Different levels of biofilm production were observed among strains. Among 48 isolates, only 4 of them (8.33%) showed strong biofilm production. By using E-test strips, amphotericin B was able to inhibit the growth of all the strains tested. Disc diffusion method used for antimicrobial sensitivity test showed that the highest activity was demonstrated by gentamicin and colistin with 95.83% (46/48) and 89.58% (43/48) of sensitive strains, respectively. Conclusions: The present study showed that RAPD-PCR was a rapid tool for discriminating P. bovis strains. Also, gentamicin and colistin can be considered as potential antimicrobial drugs which can prevent the growth of the mentioned strains in vitro, although there is no effective clinical treatment yet. Further studies are needed in order to detect an effective clinical therapy considering biofilm production by Prototheca spp. and their probable role in Prototheca pathogenicity. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract Protothecosis is a rare disease caused by achlorophilic algae of the genus Prototheca spp. In general, three clinical forms are observed: cutaneous, articular and systemic. The cutaneous form is the most common one. This study describes a patient with isolated erythematous papules and erythematous papular plaques in the scapular regions, with a previous histopathological diagnosis of cryptococcosis. New tests were conclusive for the diagnosis of protothecosis, caused by Prototheca wickerhamii.

Human disseminated protothecosis is a rare infection caused by members of the genus Prototheca , an achlorophyllic algae always associated with debilitated hosts. The presence of non-budding cells and large, spherical cells (sporangia) with endosporulation (morula) in histology is proof of Prototheca infection. Regrettably, due to the lack of specificity of clinical features and low awareness among clinicians, protothecosis is always underestimated and misdiagnosed. The available data on a species-specific analysis of this infection are limited. In this review, we summarize the etiological, epidemiological, and clinical aspects of disseminated protothecosis. The potential pathogenicity and clinical differences between P. zopfii and P. wickerhamii were observed. Additionally, the skin not only became the main invasion site but also the most involved organ by the pathogen. With the increasing numbers of immunocompromised individuals throughout the world, the incidence of disseminated infection caused by Prototheca is bound to increase, and disseminated protothecosis that accompanies skin symptoms should be taken into account by clinicians. [ABSTRACT FROM AUTHOR]

Prototheca microalgae were only recognized as pathogens of both humans and animals in the 1960s; however, since then, these microbes have been drawing increasing interest in both human and veterinary medicine. The first human outbreak of protothecosis in a tertiary care chemotherapy ward in 2018 further highlighted the need to understand in more depth and detail their ecology, etiology, pathogenesis and routes of transmission between different hosts, environments and habitats from a One Health perspective. Protothecal infections have been reported in a growing number of cattle herds around the world in recent decades, and Prototheca has become an important bovine mastitis pathogen in certain countries and regions. The survival of Prototheca in the environment and its ability to spread in the herd pose a serious challenge to the management of infected dairy farms. Prevention of the disease is particularly important, as there is no effective and reliable treatment for it and the chances of self-healing are minimal. Therefore, the development of more effective drugs is needed for the treatment of human and animal protothecosis. The prudent use of antibiotics and their replacement by alternative or preventive measures, when possible, may further contribute to the control of protothecal infections. [ABSTRACT FROM AUTHOR]

In this study, we investigated associations among subclinical intra-mammary infection (IMI) and quarter-level milk composition, udder health indicators, and cheesemaking traits. The dataset included records from 450 Holstein cows belonging to three dairy herds. After an initial screening (T0) to identify animals infected by Streptococcus agalactiae , Streptococcus uberis , Staphylococcus aureus , and Prototheca spp., 613 quarter milk samples for 2 different sampling times (T1 and T2, 1 mo after T1) were used for analysis. Milk traits were analyzed using a hierarchical linear mixed model including the effects of days in milk, parity and herd, and bacteriological and inflammatory category [culture negative with somatic cell count (SCC) <200,000 cells/mL; culture negative with SCC ≥200,000 cells/mL; or culture positive]. All udder health indicators were associated with increased SCC and IMI at both sampling times. The largest effects were detected at T2 for milk lactose (−7% and −5%) and milk conductivity (+9% and +8%). In contrast, the increase in differential SCC (DSCC) in samples with elevated SCC was larger at T1 (+17%). Culture-negative samples with SCC ≥200,000 cells/mL had the highest SCC and greatest numbers of polymorphonuclear-neutrophils-lymphocytes and macrophages at both T1 and T2. Regarding milk cheesemaking ability, samples with elevated SCC showed the worst pattern of curd firmness at T1 and T2. At T2, increased SCC and IMI induced large decreases in recoveries of nutrients into the curd, in particular recovered protein (−14% and −16%) and recovered fat (−12% and −14%). Different behaviors were observed between Strep. agalactiae and Prototheca spp., especially at T2. In particular, samples that were positive for Strep. agalactiae had higher proportions of DSCC (+19%) compared with negative samples with low SCC, whereas samples that were positive for Prototheca spp. had lower DSCC (−11%). Intramammary infection with Prototheca spp. increased milk pH compared with culture-negative samples (+3%) and negative samples that had increased SCC (+2%). The greatest impairment in curd firmness at 30 min from rennet addition was observed for samples that were positive for Prototheca spp. (−99% compared with negative samples, and −98% compared with negative samples with high SCC). These results suggest that IMI caused by Prototheca spp. have detrimental effects on milk technological traits that deserve further investigation of the mechanisms underlying animals' responses to infection. [ABSTRACT FROM AUTHOR]

BACKGROUND: Prototheca spp. belongs to the 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 milk tank Prototheca Spp prevalence in industrial dairy cattle herds in different area of Iran. And (2). To evaluate the fungal agent prevalence of bulk milk tank of dairy cattle herds in different seasons. METHODS: Bulk milk tank for the presence of Prototheca and fungal agents from 165 industrial dairy cattle herds complexes were tested. The population of each herd was 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 were 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 milk tanks of industrial dairy cattle herds in different provinces of Iran. CONCLUSIONS: 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. [ABSTRACT FROM AUTHOR]

Human disseminated protothecosis is a rare infection caused by members of the genus Prototheca, an achlorophyllic algae always associated with debilitated hosts. The presence of non-budding cells and large, spherical cells (sporangia) with endosporulation (morula) in histology is proof of Prototheca infection. Regrettably, due to the lack of specificity of clinical features and low awareness among clinicians, protothecosis is always underestimated and misdiagnosed. The available data on a species-specific analysis of this infection are limited. In this review, we summarize the etiological, epidemiological, and clinical aspects of disseminated protothecosis. The potential pathogenicity and clinical differences between P. zopfii and P. wickerhamii were observed. Additionally, the skin not only became the main invasion site but also the most involved organ by the pathogen. With the increasing numbers of immunocompromised individuals throughout the world, the incidence of disseminated infection caused by Prototheca is bound to increase, and disseminated protothecosis that accompanies skin symptoms should be taken into account by clinicians.

The majority of the nearly 10,000 described species of green algae are photoautotrophs; however, some species have lost their ability to photosynthesize and become obligate heterotrophs that rely on parasitism for survival. Two high-quality genomes of the heterotrophic algae Prototheca zopfii Pz20 and Pz23 were obtained using short- and long-read genomic as well as transcriptomic data. The genome sizes were 31.2 Mb and 31.3 Mb, respectively, and contig N50 values of 1.99 Mb and 1.26 Mb. Although P. zopfii maintained its plastid genome, the transition to heterotrophy led to a reduction in both plastid and nuclear genome size, including the loss of photosynthesis-related genes from both the nuclear and plastid genomes and the elimination of genes encoding for carotenoid oxygenase and pheophorbide an oxygenase. The loss of genes, including basic leucine-zipper (bZIP) transcription factors, flavin adenine dinucleotide-linked oxidase, and helicase, could have played a role in the transmission of autotrophy to heterotrophs and in the processes of abiotic stress resistance and pathogenicity. A total of 66 (1.37%) and 73 (1.49%) genes were identified as potential horizontal gene transfer events in the two P. zopfii genomes, respectively. Genes for malate synthase and isocitrate lyase, which are horizontally transferred from bacteria, may play a pivotal role in carbon and nitrogen metabolism as well as the pathogenicity of Prototheca and non-photosynthetic organisms. The two high-quality P. zopfii genomes provide new insights into their evolution as obligate heterotrophs and pathogenicity., Importance: The genus Prototheca , characterized by its heterotrophic nature and pathogenicity, serves as an exemplary model for investigating pathobiology. The limited understanding of the protothecosis infectious disease is attributed to the lack of genomic resources. Using HiFi long-read sequencing, both nuclear and plastid genomes were generated for two strains of P. zopfii . The findings revealed a concurrent reduction in both plastid and nuclear genome size, accompanied by the loss of genes associated with photosynthesis, carotenoid oxygenase, basic leucine-zipper (bZIP) transcription factors, and others. The analysis of horizontal gene transfer revealed the presence of 1.37% and 1.49% bacterial genes, including malate synthase and isocitrate lyase, which play crucial roles in carbon and nitrogen metabolism, as well as pathogenicity and obligate heterotrophy. The two high-quality P. zopfii genomes represent valuable resources for investigating their adaptation and evolution as obligate heterotrophs, as well as for developing future prevention and treatment strategies against protothecosis., Competing Interests: The authors declare no conflict of interest.

Background: Prototheca spp. are common and found in various environments, including animal and human intestines, on the skin and in respiratory tissues, and colonizing fingernails. Few strains pathogenic for humans have been discovered. Here, we describe an infection by the pathogenic fungus species Prototheca zopfii in a patient. The infection was initially classified as a fungus based on colony morphology, fungal staining results, and growth in some fungi culture media (Sabouraud dextrose agar [SDA]). Reports of Prototheca spp. infections are increasing, often with poor outcomes. The use of matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) technique for identification has been widely described. Phenotypic identification depends on microscopic examination of the direct wet mount and after subculturing in blood and SDA using different stains that show a typical morphological characteristic of Prototheca spp. Case Report: A 48-year-old woman was diagnosed with a P. zopfii infection after 22 days of hospitalization in the critical care unit. The patient had profound febrile neutropenia and absolute neutrophil count (ANC) was zero, associated with hypotension and disseminated intravascular coagulation (DIC) 10 days after receiving the first cycle of chemotherapy for metastatic breast adenocarcinoma. Unfortunately, the patient died within 2 days of the initiation of treatment with amphotericin B. Conclusions: This case report highlights algae infections as a possible opportunistic infection type in patients with profound neutropenia, and we discuss the use of MALDI-TOF MS-based technology in detecting such infections and predicting poor prognosis, especially in patients with the disseminated form with underlying febrile neutropenia. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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Prototheca microalgae were only recognized as pathogens of both humans and animals in the 1960s; however, since then, these microbes have been drawing increasing interest in both human and veterinary medicine. The first human outbreak of protothecosis in a tertiary care chemotherapy ward in 2018 further highlighted the need to understand in more depth and detail their ecology, etiology, pathogenesis and routes of transmission between different hosts, environments and habitats from a One Health perspective. Protothecal infections have been reported in a growing number of cattle herds around the world in recent decades, and Prototheca has become an important bovine mastitis pathogen in certain countries and regions. The survival of Prototheca in the environment and its ability to spread in the herd pose a serious challenge to the management of infected dairy farms. Prevention of the disease is particularly important, as there is no effective and reliable treatment for it and the chances of self-healing are minimal. Therefore, the development of more effective drugs is needed for the treatment of human and animal protothecosis. The prudent use of antibiotics and their replacement by alternative or preventive measures, when possible, may further contribute to the control of protothecal infections.

Abstract Background The complex cell wall structure of algae often precludes efficient extraction of their genetic material. The purpose of this study was to design a next-generation sequencing-suitable DNA isolation method for unicellular, achlorophyllous, yeast-like microalgae of the genus Prototheca, the only known plant pathogens of both humans and animals. The effectiveness of the newly proposed scheme was compared with five other, previously described methods, commonly used for DNA isolation from plants and/or yeasts, available either as laboratory-developed, in-house assays, based on liquid nitrogen grinding or different enzymatic digestion, or as commercially manufactured kits. Results All five, previously described, isolation assays yielded DNA concentrations lower than those obtained with the new method, averaging 16.15 ± 25.39 vs 74.2 ± 0.56 ng/µL, respectively. The new method was also superior in terms of DNA purity, as measured by A260/A280 (−0.41 ± 4.26 vs 2.02 ± 0.03), and A260/A230 (1.20 ± 1.12 vs 1.97 ± 0.07) ratios. Only the liquid nitrogen-based method yielded DNA of comparable quantity (60.96 ± 0.16 ng/µL) and quality (A260/A280 = 2.08 ± 0.02; A260/A230 = 2.23 ± 0.26). Still, the new method showed higher integrity, which was best illustrated upon electrophoretic analysis. Genomic DNA of Prototheca wickerhamii POL-1 strain isolated with the protocol herein proposed was successfully sequenced on the Illumina MiSeq platform. Conclusions A new method for DNA isolation from Prototheca algae is described. The method, whose protocol involves glass beads pulverization and cesium chloride (CsCl) density gradient centrifugation, was demonstrated superior over the other common assays in terms of DNA quantity and quality. The method is also the first to offer the possibility of preparation of DNA template suitable for whole genome sequencing of Prototheca spp.

Mastitis is one of the most significant diseases in dairy cows and causes several economic losses. Somatic cell count (SCC) is often used as an indirect diagnostic tool for mastitis, especially for subclinical mastitis (SCM) where no symptoms or signs can be detected. Streptococcus agalactiae is one of the main causes of contagious mastitis, and Prototheca spp. is an alga-inducing environmental mastitis that is not always correlated with increased milk SCC. The aim of this study was to evaluate the changes in the metabolomic profile of blood in relation to subclinical intramammary infection (IMI) in dairy cows. In addition, differences resulting from the etiologic agent causing mastitis were also considered. Forty Holstein-Friesian dairy cows in mid and late lactation were enrolled in this cross-sectional design study. Based on the bacteriological examination of milk, the animals were divided into 3 groups: group CTR (control group; n = 16), group A (affected by SCM with IMI caused by Strep. agalactiae; n = 17), and group P (affected by SCM with IMI caused by Prototheca spp.; n = 7). Blood samples from the jugular vein were collected in tubes containing clot activator; the serum aliquot was stored until metabolomic analysis by 1 H-nuclear magnetic resonance spectroscopy. Statistical analysis was conducted by fitting a linear model with the group as the fixed effect and SCC as the covariate. Forty-two metabolites were identified, and among them 10 were significantly different among groups. Groups A and P showed greater levels of His and lactose and lower levels of acetate, Asn, and dimethylamine compared with group CTR. Group A showed high levels of Val, and group P showed high levels of Cit and methylguanidine, as well as lower levels of 3-hydroxybutyrate, acetone, allantoin, carnitine, citrate, and ethanol. These metabolites were related to ruminal fermentations, energy metabolism, urea synthesis and metabolism, immune and inflammatory response, and mammary gland permeability. These results suggest systemic involvement with subclinical IMI and that the metabolic profile of animals with SCM undergoes changes related to the etiological agent of mastitis., (The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

A 77-year-old female patient presented with bilateral tender, swollen, erythematous, focally crusted cheeks with a discharge of pus and necrotic material, which had developed one month after autologous fat transfer and a corrective injection procedure conducted to correct an overdone fat transfer. Histopathological examination of the discharged material using routine hematoxylin and eosin staining revealed degenerated collagen admixed with three different filler materials. Scattered all throughout the specimen were numerous thick-walled, basophilic, nonbudding spherical bodies of variable sizes. The bodies stained positively with Periodic acid-Schiff and Alcian blue and showed internal septation and sporangia with a morula-like appearance. The morphology of these bodies was characteristic of a Prototheca infection. The patient was treated by surgical drainage accompanied by itraconazole 200mg daily for six months, ultimately showing marked improvement. [ABSTRACT FROM AUTHOR]

Prototheca zopfii associated with bovine mastitis and human protothecosis exists as two genotypes, of which genotype 1 is considered as non-infectious and genotype 2 as infectious. The mechanism of infection has not yet been described. The present study was aimed to identify genotype 2-specific immunodominant proteins. Prototheca proteins were separated using two-dimensional gel electrophoresis. Subsequent western blotting with rabbit hyperimmune serum revealed 28 protein spots. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis resulted in the identification of 15 proteins including malate dehydrogenase, elongation factor 1-alpha, heat shock protein 70, and 14-3-3 protein, which were previously described as immunogenic proteins of other eukaryotic pathogens.

A case of protothecosis causing endocarditis and septic shock in a dog is described. A four year-old male Labrador retriever was referred to the hospital with a history of two years of chronic diarrhea and several weeks of vestibular signs. The dog was started on antibiotic treatment but continued to deteriorate and was again presented within 3 weeks with multifocal neurological signs involving the central vestibular system and the forebrain. Several hours after admission to the hospital intensive care unit, the dog died due to cardiac arrest. Post mortem examination revealed infective endocarditis and myocarditis with the presence of prototheca species, and disseminated protothecosis. To the best knowledge of the authors this is the first report of disseminated prototheca infection in Israel in dogs. [ABSTRACT FROM AUTHOR]

Protothecosis is a rare infection caused by algae of the Prototheca genus. Its diagnosis is based on microbiological studies and on the identification in biopsies of the characteristic structures of the algae. We present the histopathological findings of protothecosis in a 72 year-old woman with the presumptive diagnosis of chronic actinic dermatitis. The pathological study of a skin biopsy revealed structures with internal septa typical of Prototheca. A review of all reports on this disease in Colombia is also presented.