Biofilm formation by Prototheca zopfii isolated from clinical and subclinical bovine mastitis in distinct growth conditions under different dyes / Formação de biofilme por Prototheca zopfii isolados de mastite bovina clínica e subclínica em distintas condições de crescimento sob diferentes corantes

ABSTRACT: Prototheca spp. have been reported as an emergent environmental mastitis pathogen in several countries. Biofilm formation is a significant factor associated with different degrees of virulence developed by many microorganisms, including Prototheca spp. The present study aimed to compare two growth conditions and two staining dyes to determine which combination was more appropriate to evaluate qualitatively and quantitatively the production of biofilm by P. zopfii. Biofilm formation was evaluated in polystyrene microplates under static and dynamic growth conditions and staining with crystal violet or cotton blue dye. All P. zopfii isolates from cows with mastitis were classified as biofilm-producers in all growth conditions and staining. The cotton blue dye proved to be more appropriate method to classify the intensity of P. zopfii biofilm production.

RESUMO: Prototheca spp. tem sido relatado como um patógeno ambiental causador de mastite bovina em vários países. A formação de biofilme é um fator associado a diferentes graus de virulência desenvolvidos por muitos microrganismos, incluindo Prototheca zopfii. O presente estudo teve como objetivo comparar duas condições de crescimento e dois corantes para determinar a combinação mais adequada para avaliar qualitativa e quantitativamente a produção de biofilme por P. zopfii. A formação de biofilme foi avaliada em microplacas de poliestireno sob condições estáticas e dinâmicas de crescimento e coloração com cristal violeta ou azul de algodão. Todos os isolados de P. zopfii de vacas com mastite foram caracterizados como produtores de biofilme, independentemente das condições de crescimento e coloração. O corante azul de algodão demonstrou ser o método mais adequado para classificar a intensidade de produção de biofilme de P. zopfii.

Onychoprotothecosis: An uncommon presentation of protothecosis.

Onychomycosis is a fairly common condition seen in a dermatology clinic. Dermatophytes Trichophyton and Epidermophyton are the known filamentous fungi implicated. The yeast‑like fungi such as Candida less commonly cause Onychomycosis. The genus Prototheca may on preliminary observation resemble yeast‑like fungi but a detailed microscopy will reveal the absence of budding and presence of endospores. Onychoprotothecosis is an uncommon presentation of human protothecosis. Of the two Prototheca species (Prototheca zopfii and Prototheca wickerhamii) known to cause the disease, P. wickerhamii has been reported more commonly. We report a culture proven case of this condition caused by P. zopfii. The patient, a 55‑year‑old housewife presented with discolouration and breaking off of the right thumb and forefinger nails since a period of six months. Samples of nail scrapping sent to the Microbiology Laboratory were culture‑positive for Prototheca. Speciation by the automated Vitek‑2 system (bioMerieux) identified the isolate as P. zopfii, which was further confirmed at PGI, Chandigarh.

Human Cutaneous Protothecosis: Report of a Case and Literature Review

The Prototheca species is achlorophyllic algae and rarely causes human infection. Human protothecosis presents clinically as a cutaneous infection, olecranon bursitis, and disseminated systemic disease. We report a case of human cutaneous protothecosis involving the left wrist. A 68-year-old man presented with an ill-defined erythematous lesion with crust at the dorsal aspect of his left wrist. A punch biopsy was performed to reveal the histologic features of granulomatous inflammation with necrosis at the upper dermis, containing Prototheca organisms, of which, the characteristic features were highlighted by special staining. Through a molecular study, the Prototheca zopfii species was identified.

Isolamento de Prototheca spp. de vacas com mastite, de leite de tanques de expansão e do ambiente dos animais / Isolation of Prototheca spp. from cows with mastitis, bulk tanks milk and in the environment of the animals

As infecções da glândula mamária de vacas leiteiras acarretam sérios prejuízos ao produtor pela diminuiçãoda produção leiteira, tratamento e descarte de animais com infecções crônicas. Elevada incidência de mastite no rebanho também altera a composição e qualidade do leite. Agentes de mastite podem causar infecções ou intoxicações no homem. Nos últimos anos tem-se dado ênfase às infecções intramamárias por algas do gênero Prototheca, agentes de infecções persistentes e refratárias à terapêutica tradicional além do caráter zoonótico. Este trabalho teve como objetivo o isolamento e identificação de Prototheca spp. de amostras de leite coletadas em tanques de expansão e latões, de vacas com mastite e do ambiente dos animais. Foram coletadas 81 amostras de leite de tanques e latões de 81 propriedades. Prototheca zopfii foi identificada em amostras de tanques de 10 propriedades. Dessas propriedades foram estudadas oito quanto à ocorrência de Prototheca spp. no ambiente do rebanho e em casos de mastite, além da identificação dos principais agentes bacterianos causadores de mastite. Foram isoladas bactérias, algas e leveduras em 324 amostras de leite provenientes de 197 vacas. Em três propriedades foi isolada P. zopfii de onze amostras de leite obtidas de cinco vacas com mastite clínica e subclínica. Nestas propriedades o agente foi isolado do meio ambiente das vacas, fezes de bezerros e teteiras. Os resultados deste trabalho mostram a importância do isolamento no tanque como indicativo da presença de Prototheca spp. em propriedades leiteiras.

The dairy cattle mammary gland infections cause serious economic losses to dairy farmers due to the decrease in milk production, therapeutic procedures and culling of chronic infected animals. Highincidence of mastitis in herds also alters both the composition and the quality of the milk. Mastitispathogens can also cause infections and poisoning in humans. In the last years, emphasis has beengiven to intramammary infections caused by the genus Prototheca which, besides their zoonotic characteristics, are considered mastitis pathogens of persistent infection and are refractory to traditional therapeutic procedures. The objective of this work was the isolation and identification of Prototheca spp.from milk samples collected from bulk tanks and milk cans, cows presenting mastitis and the dairy herdenvironment. Milk samples were collected from 81 bulk tanks and milk cans of 81 dairy herds. Protothecazopfii was identified in milk samples in 10 dairy herds. From these, eight dairy herds were studiedregarding Prototheca spp. mastitis and environmental occurrence as well as the main mastitis bacterialagents. Bacteria, algae and yeasts were isolated from 324 milk samples from 197 cows. P. zopfii wasisolated in three dairy herds from eleven milk samples from five cows with clinical and subclinical mastitis. In these dairy herds with positive isolation of P. zopfii the agent was isolated from the herd environment, excrements of the calves and teat cup rubbers. The results of this work demonstrate the importance of isolation in bulk tanks as an indicative of Prototheca spp. presence in dairy herds

Identification of a strain Prototheca zopfii var. hydrocarbonea by analyzing the sequence of ribosome RNA gene / 中国人兽共患病学报

A strain of Prototheca species isolated from a case of meningitis was identified by routine morphologic and biochemical methods as well as amplification of the related genes, in which the 28S large-subunit (LSU) region of ribosomal RNA (rRNA) gene and intergenic space (ITS) were amplified with universal fungal primers. The small-subunit (SSU) rRNA gene was amplified with eukaryote-specific primers and Prototheca genus-specific primers. Then, compared the sequences with the ones posted on BLAST (www. ncbi. nlm. nih. gov/BLAST). The organism choice giving the closest match, up to 99%, was considered the most likely correct identification. It was found that this strain of fungus grew well at 25 ℃ or 37 ℃. Smooth,moist colonies with white color were observed on Sabouraud Dextrose Agar (SDA) and Potato Dextrose Agar (PDA). Microscopically, globular or ovoid cells, a number of round, ovoid shaped endospores could be observed. No hypha, ascus or blastic conidia was found upon cultivation on SDA. Based on the morphological characteristics, this isolate could be identified as Prototheca species. The identity with Prototheca wickerhamii was 2.9 % as demonstrated by the API 20C AUX system. Sequence analysis showed that the ITS gene was proved to be a complex structural region which was not suitable for the identification of Prototheca species, but the LSU and SSU rDNA regions showed 94% and 99.9% sequence identities with Prototheca zopfii var. hydrocarbonea (P. zopfii var. hydrocarbonea) respectively, indicating that the SSU rRNA gene sequence might be more reliable on than the LSU rRNA gene sequence for identification of Prototheca species.