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 15.625 µg ml-1 to 62.5 µg 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.

First bloodstream infection due to Prototheca zopfii var. hydrocarbonea in an immunocompromised patient.

Here we describe a bloodstream infection due to P. zopfii var. hydrocarbonea in a patient with acute lymphoblastic leukemia. Identification was performed by DNA sequencing of the D1/D2 domain of 26s ribosomal DNA and by MALDI-TOF MS technique. Antifungal susceptibility tests against amphotericin B, fluconazole, itraconazole, and voriconazole showed the following MIC values, respectively: 0.25 mg/L, 128 mg/L, 0.064 mg/L, and 0.125 mg/L. The patient received amphotericin B treatment with a successful outcome.

Systemic canine protothecosis

Protothecosis is a rare disease caused by achlorophyllic algae of the genus Prototheca. This case involved a 5 year-old intact female Cocker Spaniel, with a history of mild and persistent diarrhea that was not responsive to treatment. Clinical signs progressed to watery and bloody diarrhea followed by development of ocular signs, including complete and bilateral retinal displacement, glaucoma, synechia, and blindness. At necropsy, lesions observed consisted of necro-hemorrhagic enteritis, and multiple white nodular lesions in the heart, pancreas, kidneys, and thyroid gland. Microscopically, the nodular lesions were characterized by a chronic histiocytic inflammatory infiltrate associated with a severe accumulation of organisms with morphology and histochemical features compatible with Prototheca sp.

Systemic canine protothecosis

Protothecosis is a rare disease caused by achlorophyllic algae of the genus Prototheca. This case involved a 5 year-old intact female Cocker Spaniel, with a history of mild and persistent diarrhea that was not responsive to treatment. Clinical signs progressed to watery and bloody diarrhea followed by development of ocular signs, including complete and bilateral retinal displacement, glaucoma, synechia, and blindness. At necropsy, lesions observed consisted of necro-hemorrhagic enteritis, and multiple white nodular lesions in the heart, pancreas, kidneys, and thyroid gland. Microscopically, the nodular lesions were characterized by a chronic histiocytic inflammatory infiltrate associated with a severe accumulation of organisms with morphology and histochemical features compatible with Prototheca sp.

Fatores de risco associados à mastite bovina causada por Prototheca zopfii / Risk factors associated with bovine mastitis caused by Prototheca zopfii

Este trabalho teve como objetivo o estudo de fatores de risco associados à mastite bovina causada por Prototheca zopfii. Foram analisadas 13 propriedades leiteiras dos Estados do Paraná e de São Paulo, segundo os seguintes critérios de seleção: confirmação prévia de casos de mastite por Prototheca spp., triagem pela pesquisa de Prototheca spp. em tanques de expansão e latões e rebanhos com contagem de células somáticas acima de 5x105cel mL-1. As amostras coletadas consistiram de: leite, água, solo, fezes e swab de teteiras. Prototheca spp. foi isolada de amostras de leite dos quartos mamários com mastite clínica ou subclínica em uma propriedade e de amostras de leite e do ambiente em quatro propriedades, nas quais foi isolada em amostras de: água de bebedouro, abastecimento, esgoto, empoçada no piso de estábulo e sala de ordenha, solo de piquete e pasto, teteiras, fezes de bezerros e suínos. Do total de 383 vacas examinadas, Prototheca spp. foi isolada em 20 (5,2 por cento) vacas, sendo caracterizada como P. zopfii em 18. Os fatores de risco associados à mastite causada por P. zopfii foram: criação das vacas a pasto, alimentação dos animais com pasto e silagem, realização de ordenha mecânica em estábulo, permanência das vacas após ordenha em piquete sem alimento, criação de suínos próxima às instalações dos bovinos, existência de cães, gatos e roedores, falta de higienização dos tetos com água, pré-imersão dos tetos em aplicador com retorno e sem a troca do anti-séptico, alimentação dos bezerros com leite de vacas com mastite clínica e serem as vacas da raça holandesa.

This research had as objective the study of risk factors associated with bovine mastitis caused by Prototheca zopfii. Thirteen dairy herds in Paraná and São Paulo states were analyzed and selected according to the following criteria: previous confirmation of Prototheca spp. mastitis cases, screening of Prototheca spp. in bulk tanks and milk cans, and herds with somatic cells count over 5x105cel mL-1. The samples collected consisted of: milk, water, soil, manure and swabs of teat cup rubbers. Prototheca spp. was isolated from mammary quarters with clinical and subclinical mastitis of milk samples in one herd and from the environment and cows in four herds. Out of 383 cows examined, Prototheca spp. was isolated in 20 (5.2 percent) cows with mastitis, and 18 of them were characterized as P. zopfii. In four herds when Prototheca spp. was identified from mammary quarters and environment the agent was isolated from the following samples: water in the waterers, puddled water in the stalls and the milking parlour, supply, sewage, cow pen and pasture soil, teat cup rubbers and manure from calves and swines. The risk factors associated with P. zopfii mastitis consisted of: pasture system, pasture and silage feeding, use of milking machine in stalls, cow pen without fresh feed after milking, raising of swines near bovine housing, existence of dogs, cats and rodents, absence of teats hygienization with water, use of pre-immersion devices with return and without change of antiseptic, calves fed with milk of clinical mastitis cases and the Holstein breed.

Fatores de risco associados à mastite bovina causada por Prototheca zopfii / Risk factors associated with bovine mastitis caused by Prototheca zopfii

Este trabalho teve como objetivo o estudo de fatores de risco associados à mastite bovina causada por Prototheca zopfii. Foram analisadas 13 propriedades leiteiras dos Estados do Paraná e de São Paulo, segundo os seguintes critérios de seleção: confirmação prévia de casos de mastite por Prototheca spp., triagem pela pesquisa de Prototheca spp. em tanques de expansão e latões e rebanhos com contagem de células somáticas acima de 5x105cel mL-1. As amostras coletadas consistiram de: leite, água, solo, fezes e swab de teteiras. Prototheca spp. foi isolada de amostras de leite dos quartos mamários com mastite clínica ou subclínica em uma propriedade e de amostras de leite e do ambiente em quatro propriedades, nas quais foi isolada em amostras de: água de bebedouro, abastecimento, esgoto, empoçada no piso de estábulo e sala de ordenha, solo de piquete e pasto, teteiras, fezes de bezerros e suínos. Do total de 383 vacas examinadas, Prototheca spp. foi isolada em 20 (5,2 por cento) vacas, sendo caracterizada como P. zopfii em 18. Os fatores de risco associados à mastite causada por P. zopfii foram: criação das vacas a pasto, alimentação dos animais com pasto e silagem, realização de ordenha mecânica em estábulo, permanência das vacas após ordenha em piquete sem alimento, criação de suínos próxima às instalações dos bovinos, existência de cães, gatos e roedores, falta de higienização dos tetos com água, pré-imersão dos tetos em aplicador com retorno e sem a troca do anti-séptico, alimentação dos bezerros com leite de vacas com mastite clínica e serem as vacas da raça holandesa.(AU)

This research had as objective the study of risk factors associated with bovine mastitis caused by Prototheca zopfii. Thirteen dairy herds in Paraná and São Paulo states were analyzed and selected according to the following criteria: previous confirmation of Prototheca spp. mastitis cases, screening of Prototheca spp. in bulk tanks and milk cans, and herds with somatic cells count over 5x105cel mL-1. The samples collected consisted of: milk, water, soil, manure and swabs of teat cup rubbers. Prototheca spp. was isolated from mammary quarters with clinical and subclinical mastitis of milk samples in one herd and from the environment and cows in four herds. Out of 383 cows examined, Prototheca spp. was isolated in 20 (5.2 percent) cows with mastitis, and 18 of them were characterized as P. zopfii. In four herds when Prototheca spp. was identified from mammary quarters and environment the agent was isolated from the following samples: water in the waterers, puddled water in the stalls and the milking parlour, supply, sewage, cow pen and pasture soil, teat cup rubbers and manure from calves and swines. The risk factors associated with P. zopfii mastitis consisted of: pasture system, pasture and silage feeding, use of milking machine in stalls, cow pen without fresh feed after milking, raising of swines near bovine housing, existence of dogs, cats and rodents, absence of teats hygienization with water, use of pre-immersion devices with return and without change of antiseptic, calves fed with milk of clinical mastitis cases and the Holstein breed.(AU)

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

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

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. High incidence of mastitis in herds also alters both the composition and the quality of the milk. Mastitis pathogens can also cause infections and poisoning in humans. In the last years, emphasis has been given 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 herd environment. Milk samples were collected from 81 bulk tanks and milk cans of 81 dairy herds. Prototheca zopfii was identified in milk samples in 10 dairy herds. From these, eight dairy herds were studied regarding Prototheca spp. mastitis and environmental occurrence as well as the main mastitis bacterial agents. Bacteria, algae and yeasts were isolated from 324 milk samples from 197 cows. P. zopfii was isolated 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

As infecções da glândula mamária de vacas leiteiras acarretam sérios prejuízos ao produtor pela diminuição da 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 bezerr

The use of successive milking as a procedure for the elimination of infection on due to Prototheca spp in cases of bovine clinical mastitis. (A case report) / Utilização de ordenhas sucessivas como procedimento para eliminação de infecção por prototheca spp de casos de mastite clínica bovina (relato de caso)

This work relates the case of two animals (Black and White Holstein) from a bovine milking herd, from north of Paraná State, in which mastitis due Prototheca spp was diagnosed. As a procedure for the infection elimination, it was opted for the use of successive milking performed six times daily, during eight days. The animals were followed up through mycological cultures from milk Samples after 15, 30 and 60 days from the beginning of the procedure. The clinical signs regressed after three days and the mycologicai cultures were negative after the 15th day.  

O presente trabalho relata um caso de dois animais HPB de um rebanho bovino leiteiro, da região norte do Estado do Paraná, dos quais diagnosticou-se mastite clínica por Prototheca spp. Como procedimento para eliminação da infecção optou-se peta utilização de ordenhas sucessivas praticadas seis vezes diariamente durante oito dias. Os animais foram acompanhados através de culturas micológicas das amostras de leite após 15, 30 e 60 dias do inicio do procedimento. Os sinais clínicos regrediram após três dias, e a partir do 15 dia as culturas foram negativas.