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Tomasz Jagielski - One of the best experts on this subject based on the ideXlab platform.
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prevalence of Prototheca spp on dairy farms in poland a cross country study
Microbial Biotechnology, 2019Co-Authors: Tomasz Jagielski, Zofia Bakula, Katarzyna Roeske, Henryk Krukowski, M Bochniarz, T Piech, łukasz Wlazlo, Piotr WochAbstract:: The Prototheca algae have recently emerged as an important cause of bovine mastitis globally. Here, we present results of a first large-scale, cross-country survey on the prevalence of Prototheca spp. in dairy cows, and their environment in Poland. A total of 1211 samples were collected and microbiologically analysed. Included within this number were milk (n = 638), body swabs (n = 374) and environmental samples (n = 199), originating from 400 dairy cows and their surroundings, on 16 dairy farms, based in all major provinces of the country. Prototheca spp. were the third, after Streptococcus and Staphylococcus spp., most common mastitis pathogens. The overall prevalence of Protothecal mastitis was 8.3% (33/400), with the majority (75.8%) of cases having a subclinical course, and all but one attributable to P. zopfii genotype 2. Prototheca spp. were cultured from body swabs of both healthy and mastitic cows, yet the isolation rate among the latter was conspicuously lower (12.3% vs. 17.8%). Forty-two (21.2%) environmental samples yielded growth of Prototheca spp. However, no clear association between Prototheca mastitis in dairy cows and the algal isolation from the herd environment was found. Nor was there any association between the environmental recovery of the algae and farm management practices.
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a survey on the incidence of Prototheca mastitis in dairy herds in lublin province poland
Journal of Dairy Science, 2019Co-Authors: Tomasz Jagielski, Zofia Bakula, Katarzyna Roeske, M Bochniarz, T Piech, łukasz Wlazlo, Piotr Woch, Henryk KrukowskiAbstract:ABSTRACT Prototheca mastitis has recently become an emerging disease; although its incidence is increasing steadily, its epidemiology remains largely understudied. The aim of this work was to investigate the prevalence of Prototheca spp. in dairy cows and their environment in Lublin province, covering most of southeastern Poland. Between December 2015 and July 2016, a total of 172 milking cows from 10 dairy farms were inspected for mastitis using clinical examination and the California Mastitis Test (CMT). Quarter milk samples (QMS, n = 179) and body site swabs (n = 151) from CMT-positive cows were collected for microbiological culture. In addition, we evaluated QMS and body site swabs from 23 healthy cows, along with 91 environmental samples. Of 100 CMT-positive cows, 71 had at least one QMS positive for microbial growth. In 8 (11.3%) of these cows, originating from 7 dairy farms, Prototheca spp. were cultured. The average somatic cell count of the Prototheca-containing milk was 4.02 × 106 cells/mL compared with 0.13 × 106 cells/mL of the Prototheca-free milk (collected from control animals). No significant differences were observed between mastitis and control cows with respect to counts of total white blood cells, lymphocytes, neutrophils, and eosinophils. Half of the cows with Prototheca spp. in their milk did not yield the algae from other anatomical sites. Eight cows were negative for the presence of Prototheca spp. in their milk but positive for the algae in swabs from anatomical sites. Among the environmental sources that were positive for Prototheca growth were watering troughs, manure, feed, and mud. All (45) Prototheca isolates recovered in this study were subjected to species- and genotype-level molecular identification. All QMS and most of the animal swabs (90%) yielded Prototheca zopfii genotype (gen.) 2. Of the animal samples, P. zopfii gen. 1 and Prototheca blaschkeae were isolated only from feces and rectum. Environmental samples grew either P. zopfii gen. 2 (67%) or P. zopfii gen. 1 (33%). This study demonstrates that P. zopfii gen. 2 is the third most common pathogen of mastitis in cattle in southeast Poland, with an overall incidence of 4.6%. Finding Prototheca spp., including P. zopfii gen. 1 and 2 and P. blaschkeae, in stool and rectal swabs from healthy animals may suggest their role as nonpathogenic microflora of bovine gut.
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cytb as a new genetic marker for differentiation of Prototheca species
Journal of Clinical Microbiology, 2018Co-Authors: Tomasz Jagielski, Jan Gawor, Zofia Bakula, Przemyslaw Decewicz, Kacper Maciszewski, Anna KarnkowskaAbstract:Achlorophyllous unicellular microalgae of the genus Prototheca (Trebouxiophyceae, Chlorophyta) are the only known plants that cause infections in both humans and animals, collectively referred to as protothecosis. Human protothecosis, most commonly manifested as cutaneous, articular, and disseminated disease, is primarily caused by Prototheca wickerhamii, followed by Prototheca zopfii and, sporadically, by Prototheca cutis and Prototheca miyajii. In veterinary medicine, however, P. zopfii is a major pathogen responsible for bovine mastitis, which is a predominant form of Protothecal disease in animals. Historically, identification of Prototheca spp. has relied upon phenotypic criteria; these were later replaced by molecular typing schemes, including DNA sequencing. However, the molecular markers interrogated so far, mostly located in the ribosomal DNA (rDNA) cluster, do not provide sufficient discriminatory power to distinguish among all Prototheca spp. currently recognized. Our study is the first attempt to develop a fast, reliable, and specific molecular method allowing identification of all Prototheca spp. We propose the mitochondrial cytb gene as a new and robust marker for diagnostics and phylogenetic studies of the Prototheca algae. The cytb gene displayed important advantages over the rDNA markers. Not only did the cytb gene have the highest discriminatory capacity for resolving all Prototheca species, but it also performed best in terms of technical feasibility, understood as ease of amplification, sequencing, and multiple alignment analysis. Based on the species-specific polymorphisms in the partial cytb gene, we developed a fast and straightforward PCR-restriction fragment length polymorphism (RFLP) assay for identification and differentiation of all Prototheca species described so far. The newly proposed method is advocated to be a new gold standard in diagnostics of Protothecal infections in human and animal populations.
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the activity of silver nanoparticles against microalgae of the Prototheca genus
Nanomedicine: Nanotechnology Biology and Medicine, 2018Co-Authors: Tomasz Jagielski, Zofia Bakula, Malgorzata Plen, Michal Kaminski, Julita Nowakowska, Jacek Bielecki, Krystyna I Wolska, Anna M GrudniakAbstract:AIM: To investigate the in vitro activity of silver NPs (AgNPs) against pathogenic microalgae of the Prototheca genus. MATERIALS & METHODS: The antialgal potential of AgNPs against Prototheca species of both clinical and environmental origin was assessed from minimum inhibitory (algistatic) and algicidal concentrations. The in vitro cytotoxicity of AgNPs against bovine mammary epithelial cell line was evaluated by means of the standard MTT assay. RESULTS: AgNPs showed a strong killing activity toward Prototheca algae, as the minimal algicidal concentration (MAC) values matched perfectly the corresponding minimum inhibitory concentration (MIC) values for all species (MAC = MIC, 1-4 mg/l), except P. stagnora (MIC > 8 mg/l). The concentrations inhibitory to pathogenic Prototheca spp. (MIC, 1-4 mg/l) were below the concentrations at which any toxicity in epithelial cells could be observed (CC20 > 6 mg/l). CONCLUSION: The study emphasizes the potential of AgNPs as a new therapeutic tool for the management of Prototheca infections.
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3 bromopyruvate as an alternative option for the treatment of protothecosis
Frontiers in Pharmacology, 2018Co-Authors: Tomasz Jagielski, Katarzyna Niedźwiecka, Katarzyna Roeske, Mariusz DylągAbstract:Protothecosis is an unusual infection of both humans and animals caused by opportunistically pathogenic microalgae of the genus Prototheca. Until now, no standardized treatment protocols exist for the Protothecal disease, boosted by a remarkable resistance of Prototheca spp. to a wide array of antimicrobial agents currently available in clinical use. Consequently, there is an urgent need for new effective drugs against Prototheca algae. In this study, the anti-Prototheca activity of 3-bromopyruvate (3BP), either alone or in combination with amphotericin B (AMB) was assessed in vitro, as well as the cytotoxicity of 3BP towards the bovine mammary epithelial cells and murine skin fibroblasts. The mean minimum inhibitory concentrations (MIC) and minimum algaecidal concentrations (MAC) were 0.85±0.21 and 2.25±0.54 mM for Prototheca wickerhamii, 1.25±0.47 and 4.8±1.03 mM for Prototheca blaschkeae, and 1.55±0.69 and 5.6±1.3 mM for Prototheca zopfii gen. 2, respectively. For all Prototheca strains tested, a synergistic interaction between 3BP and AMB was observed, resulting in about 4-fold reduction of their individual MICs, when used together. The elevated content of intracellular glutathione (GSH) was associated with a decreased susceptibility to 3BP. Both epithelial and fibroblast cells retained high viability upon treatment with 3BP at concentrations equivalent to the highest MIC recorded (3 mM) and 10-fold higher (30 mM), with the mean cell viability exceeding 80%, essentially the same as for the untreated cells. The results from these in vitro studies emphasize the high activity of 3BP against the Prototheca algae, its synergistic effect when used in combination with AMB, and the safety of the drug towards the tested mammalian cells. Along with the advantageous physico-chemical and pharmacokinetic properties, 3BP may be considered an effective and safe novel agent against the Protothecal disease.
Uwe Roesler - One of the best experts on this subject based on the ideXlab platform.
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multicentre etest evaluation of in vitro activity of conventional antifungal drugs against european bovine mastitis Prototheca spp isolates
Journal of Antimicrobial Chemotherapy, 2012Co-Authors: Tomasz Jagielski, P E Lagneau, Eva Branda, Benedetta Turchetti, Pietro Buzzini, Uwe Roesler, H Lassa, Edward Malinowski, Angela Polleichtner, Sara MarquesAbstract:OBJECTIVES: Bovine mammary protothecosis is a serious pathology that entails high economic losses in the dairy industry. The disease, the frequency of which has recently been increasing worldwide, is caused by unicellular, achlorophyllous, yeast-like algae of two species: Prototheca zopfii and Prototheca blaschkeae. The objective of this study was to investigate the in vitro activity of a panel of conventional antifungal drugs against Prototheca spp. isolates. METHODS: A total of 144 P. zopfii genotype 2 and P. blaschkeae strains isolated from milk of mastitic cows were subjected to drug susceptibility testing by Etest methodology. RESULTS: Five out of ten antifungal drugs tested exhibited no activity against Prototheca spp. isolates. The best activity against Prototheca spp. was demonstrated by amphotericin B (MIC₉₀ of 1.5 mg/L). The MICs differed significantly (P < 0.01) between P. zopfii genotype 2 and P. blaschkeae, with the latter species being more susceptible to amphotericin B and azoles. Marked differences (P < 0.05) in azole and amphotericin B activities were noted among Prototheca spp. isolates originating from different European countries. Based on the correlation coefficients, a considerable cross-interaction was found among MICs of azoles and between MICs of azoles and amphotericin B for Prototheca spp. (P < 0.03). CONCLUSIONS: This study represents the largest, cross-European evaluation of antifungal activity against Prototheca spp. to date. The activity of amphotericin B against Prototheca spp. validates its potential use as a therapeutic agent against bovine protothecosis. For laboratory testing of drug activity against Prototheca spp., the Etest method is encouraged, due to its technical simplicity, rapidity and high intra- and inter-laboratory reproducibility.
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establishment of a matrix assisted laser desorption ionization time of flight mass spectrometry database for rapid identification of infectious achlorophyllous green micro algae of the genus Prototheca
Clinical Microbiology and Infection, 2012Co-Authors: Jayaseelan Murugaiyan, Jennifer Ahrholdt, V Kowbel, Uwe RoeslerAbstract:Abstract The possibility of using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) for rapid identification of pathogenic and non-pathogenic species of the genus Prototheca has been recently demonstrated. A unique reference database of MALDI-TOF MS profiles for type and reference strains of the six generally accepted Prototheca species was established. The database quality was reinforced after the acquisition of 27 spectra for selected Prototheca strains, with three biological and technical replicates for each of 18 type and reference strains of Prototheca and four strains of Chlorella . This provides reproducible and unique spectra covering a wide m/z range (2000–20 000 Da) for each of the strains used in the present study. The reproducibility of the spectra was further confirmed by employing composite correlation index calculation and main spectra library (MSP) dendrogram creation, available with MALDI Biotyper software. The MSP dendrograms obtained were comparable with the 18S rDNA sequence-based dendrograms. These reference spectra were successfully added to the Bruker database, and the efficiency of identification was evaluated by cross-reference-based and unknown Prototheca identification. It is proposed that the addition of further strains would reinforce the reference spectra library for rapid identification of Prototheca strains to the genus and species/genotype level.
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epidemiological analysis of worldwide bovine canine and human clinical Prototheca isolates by pcr genotyping and maldi tof mass spectrometry proteomic phenotyping
Medical Mycology, 2012Co-Authors: Jennifer Ahrholdt, Tomasz Jagielski, Jayaseelan Murugaiyan, Reinhard K Straubinger, Uwe RoeslerAbstract:This study presents information on the phenotypic and genotypic characterization of clinical Prototheca spp. isolates obtained from different geographic regions. Of 350 isolates studied, 342 came from cattle, six from canines and two from humans. Phenotypic characterization was carried out by a matrix-assisted laser desorption/ionization-time of fl ight (MALDI-TOF) proteomic analysis. The peptide extraction that was used for this analysis included the additional steps of washing and sonication to increase the yield of peptide. Genotypic analysis was conducted using species- and genotype-specifi c primers. The study revealed that among the cattle isolates, 310 (90.6%) belonged to Prototheca zopfi i genotype 2, 30 (8.8%) to P. blaschkeae , and two (0.6%) to P. zopfi i genotype 1. P. zopfi i genotype 2 is the principal etiological agent of Protothecal mastitis in cattle regardless of the geographic region. Similarly, all canine and human isolates also belonged to the P. zopfi i genotype 2, suggesting that this is probably the most virulent species of the genus. The role of P. blaschkeae needs further epidemiologic studies to ascertain its etiologic role in bovine mastitis. To the best of our knowledge, this is the fi rst comprehensive study on phenotypic and genotypic characterization of P. zopfi i and P. blaschkeae isolates originating from diverse clinical specimens from different countries.
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genotyping of bovine Prototheca mastitis isolates from poland
Veterinary Microbiology, 2011Co-Authors: Tomasz Jagielski, H Lassa, Jennifer Ahrholdt, Edward Malinowski, Uwe RoeslerAbstract:Abstract Bovine mastitis due to unicellular, achlorophyllous algae of the genus Prototheca is a serious and complex ailment that accounts for high economic losses in the dairy industry. Bovine Protothecal mastitis has been almost exclusively associated with only one species Prototheca zopfii . Recently, based on the 18S rDNA sequence analysis, P. zopfii has been divided into three genotypes (1–3), of which P. zopfii genotype 3 has been given the status of a new species Prototheca blaschkeae sp. nov. The aim of this study was to investigate the genotypic composition of the population of P. zopfii bovine mastitis isolates from Poland, according to that threefold classification. Of the 44 isolates tested, 43 (98%) were identified as P. zopfii genotype 2. Only one isolate was assigned to the newly established P. blaschkeae species. These findings clearly show the predominance of the P. zopfii genotype 2 in the etiology of bovine mammary protothecosis in Poland, but also underline the involvement of P. blaschkeae in the disease. The study is the first to provide molecular characterization of Polish P. zopfii mastitis isolates, collected from different regions of Poland.
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Prototheca zopfii genotype 2 the causative agent of bovine Protothecal mastitis
Veterinary Microbiology, 2007Co-Authors: Asja Moller, Uwe Truyen, Uwe RoeslerAbstract:In order to clarify the epidemiology of bovine Protothecal mastitis, 30 Prototheca zopfii mastitis isolates were genetically investigated. Based on the 18S rDNA, which allows a differentiation of the former species P. zopfii in two distinct P. zopfii genotypes and Prototheca blaschkeae sp. nov., newly developed genotype-specific PCR-assays as well as RFLP-assays were applied. All mastitis isolates investigated could be assigned to P. zopfii genotype 2 suggesting that this genotype is the aetiological agent of bovine Prototheca mastitis.
Sara Marques - One of the best experts on this subject based on the ideXlab platform.
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internal transcribed spacer sequence based rapid molecular identification of Prototheca zopfii and Prototheca blaschkeae directly from milk of infected cows
Journal of Dairy Science, 2015Co-Authors: Sara Marques, Volker A R Huss, K Pfisterer, C Grosse, Gertrude ThompsonAbstract:The increasing incidence of rare mastitis-causing pathogens has urged the implementation of fast and efficient diagnostic and control measures. Prototheca algae are known to be associated with diseases in humans and animals. In the latter, the most prevalent form of protothecosis is bovine mastitis with Prototheca zopfii and Prototheca blaschkeae representing the most common pathogenic species. These nonphotosynthetic and colorless green algae are ubiquitous in different environments and are widely resistant against harmful conditions and antimicrobials. Hence, the association of Prototheca with bovine mastitis represents a herd problem, requiring fast and easy identification of the infectious agent. The purpose of this study was to develop a reliable and rapid method, based on the internal transcribed spacer (ITS) sequences of ribosomal DNA, for molecular identification and discrimination between P. zopfii and P. blaschkeae in bovine mastitic milk. The complete ITS sequences of 32 Prototheca isolates showed substantial interspecies but moderate intraspecies variability facilitating the design of species-specific PCR amplification primers. The species-specific PCR was successfully applied to the identification of P. zopfii and P. blaschkeae directly from milk samples. The intraspecific ITS phylogeny was compared for each species with the geographical distribution of the respective Prototheca isolates, but no significant correlation was found.
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multicentre etest evaluation of in vitro activity of conventional antifungal drugs against european bovine mastitis Prototheca spp isolates
Journal of Antimicrobial Chemotherapy, 2012Co-Authors: Tomasz Jagielski, P E Lagneau, Eva Branda, Benedetta Turchetti, Pietro Buzzini, Uwe Roesler, H Lassa, Edward Malinowski, Angela Polleichtner, Sara MarquesAbstract:OBJECTIVES: Bovine mammary protothecosis is a serious pathology that entails high economic losses in the dairy industry. The disease, the frequency of which has recently been increasing worldwide, is caused by unicellular, achlorophyllous, yeast-like algae of two species: Prototheca zopfii and Prototheca blaschkeae. The objective of this study was to investigate the in vitro activity of a panel of conventional antifungal drugs against Prototheca spp. isolates. METHODS: A total of 144 P. zopfii genotype 2 and P. blaschkeae strains isolated from milk of mastitic cows were subjected to drug susceptibility testing by Etest methodology. RESULTS: Five out of ten antifungal drugs tested exhibited no activity against Prototheca spp. isolates. The best activity against Prototheca spp. was demonstrated by amphotericin B (MIC₉₀ of 1.5 mg/L). The MICs differed significantly (P < 0.01) between P. zopfii genotype 2 and P. blaschkeae, with the latter species being more susceptible to amphotericin B and azoles. Marked differences (P < 0.05) in azole and amphotericin B activities were noted among Prototheca spp. isolates originating from different European countries. Based on the correlation coefficients, a considerable cross-interaction was found among MICs of azoles and between MICs of azoles and amphotericin B for Prototheca spp. (P < 0.03). CONCLUSIONS: This study represents the largest, cross-European evaluation of antifungal activity against Prototheca spp. to date. The activity of amphotericin B against Prototheca spp. validates its potential use as a therapeutic agent against bovine protothecosis. For laboratory testing of drug activity against Prototheca spp., the Etest method is encouraged, due to its technical simplicity, rapidity and high intra- and inter-laboratory reproducibility.
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phenotypic characterization of mastitic Prototheca spp isolates
Research in Veterinary Science, 2010Co-Authors: Sara Marques, Eliane Silva, J Carvalheira, Gertrude ThompsonAbstract:Abstract Bovine mastitis associated with Prototheca is considered a rare pathology, but is increasing in prevalence all over the world and therefore becoming more relevant to the dairy industry. The biochemical characterization of 47 Prototheca isolates retrieved from mastitic milk was performed in this study using API 20C Aux and two BBL Crystal Kits, followed by an analysis with InforBio software. The usage of this methodology, allowed the identification of discriminative phenotypic characteristics for the strains tested. The differential-character-finding algorithm used by this software permitted the identification of new phenotypic characteristics to discriminate between Prototheca zopfii, P. blaschkeae and P. wickerhamii, such as, citrate, phosphorycholine and arabinoside. The main objective of this study was to determine new phenotypic characteristics that allowed a better characterization of Prototheca spp. Usage of recent bioinformatic tools improved the analyses of several features that are important for a better characterization of Prototheca spp.
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bovine mastitis associated with Prototheca blaschkeae
Journal of Clinical Microbiology, 2008Co-Authors: Sara Marques, Eliane Silva, J Carvalheira, Volker A R Huss, Christine Kraft, Arnaldo Videira, Gertrude ThompsonAbstract:Bovine mastitis is an important and complex disease responsible for economic losses in the dairy industry. Biotype II strains of the green alga Prototheca zopfii can be involved, most often resulting in chronic mastitis of difficult treatment associated with reduced milk production. This type of infection is rare, but the number of reported cases is increasing worldwide. In order to determine the kind of species involved in mastitis by Prototheca in northwest Portugal, 41 Prototheca isolates were genetically characterized. The algae are part of Prototheca isolates that were collected during a 6-year period, isolated from the milk of 41 dairy cows in a total of 22 herds with a history of increasing somatic cell counts, mild clinical signs of udder infection, and unsuccessful response to the usual therapy. PCR amplification of the 18S ribosomal DNA (rDNA), amplified rDNA restriction analysis, and phylogenetic analyses of the 18S rDNA sequences were performed. Thirty-seven isolates were identified as P. zopfii var. hydrocarbonea and four as Prototheca blaschkeae. These data suggest a high incidence of P. zopfii var. hydrocarbonea mastitis in the region and demonstrate for the first time the involvement of P. blaschkeae with bovine mammary gland infections.
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short communication in vitro antimicrobial susceptibility of Prototheca wickerhamii and Prototheca zopfii isolated from bovine mastitis
Journal of Dairy Science, 2006Co-Authors: Sara Marques, Eliane Silva, J Carvalheira, Gertrude ThompsonAbstract:Bovine mastitis caused by Prototheca spp. can assume high significance because of economic losses and the potential risk to public health. Studies on the susceptibility of Prototheca spp. to antimicrobials have demonstrated its high level of resistance. We report the susceptibility of bovine isolates of Prototheca wickerhamii and Prototheca zopfii to amphotericin B and nystatin, 2 antifungal agents commonly used in the control of protothecosis, and discuss the results. After subculture, minimum inhibitory concentrations of both antifungal drugs were determined using macrodilution and agar diffusion methods. The inoculum concentration was standardized by determination of colony-forming units per milliliter. Nystatin showed more efficacy than amphotericin B in inhibiting P. wickerhamii growth. In contrast, growth inhibition of P. zopfii was similar for both antifungal agents. This study demonstrates different in vitro susceptibility patterns of P. wickerhamii and P. zopfii, reinforcing the necessity for more investigation into drugs that can be used with clinical efficacy.
Gertrude Thompson - One of the best experts on this subject based on the ideXlab platform.
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internal transcribed spacer sequence based rapid molecular identification of Prototheca zopfii and Prototheca blaschkeae directly from milk of infected cows
Journal of Dairy Science, 2015Co-Authors: Sara Marques, Volker A R Huss, K Pfisterer, C Grosse, Gertrude ThompsonAbstract:The increasing incidence of rare mastitis-causing pathogens has urged the implementation of fast and efficient diagnostic and control measures. Prototheca algae are known to be associated with diseases in humans and animals. In the latter, the most prevalent form of protothecosis is bovine mastitis with Prototheca zopfii and Prototheca blaschkeae representing the most common pathogenic species. These nonphotosynthetic and colorless green algae are ubiquitous in different environments and are widely resistant against harmful conditions and antimicrobials. Hence, the association of Prototheca with bovine mastitis represents a herd problem, requiring fast and easy identification of the infectious agent. The purpose of this study was to develop a reliable and rapid method, based on the internal transcribed spacer (ITS) sequences of ribosomal DNA, for molecular identification and discrimination between P. zopfii and P. blaschkeae in bovine mastitic milk. The complete ITS sequences of 32 Prototheca isolates showed substantial interspecies but moderate intraspecies variability facilitating the design of species-specific PCR amplification primers. The species-specific PCR was successfully applied to the identification of P. zopfii and P. blaschkeae directly from milk samples. The intraspecific ITS phylogeny was compared for each species with the geographical distribution of the respective Prototheca isolates, but no significant correlation was found.
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phenotypic characterization of mastitic Prototheca spp isolates
Research in Veterinary Science, 2010Co-Authors: Sara Marques, Eliane Silva, J Carvalheira, Gertrude ThompsonAbstract:Abstract Bovine mastitis associated with Prototheca is considered a rare pathology, but is increasing in prevalence all over the world and therefore becoming more relevant to the dairy industry. The biochemical characterization of 47 Prototheca isolates retrieved from mastitic milk was performed in this study using API 20C Aux and two BBL Crystal Kits, followed by an analysis with InforBio software. The usage of this methodology, allowed the identification of discriminative phenotypic characteristics for the strains tested. The differential-character-finding algorithm used by this software permitted the identification of new phenotypic characteristics to discriminate between Prototheca zopfii, P. blaschkeae and P. wickerhamii, such as, citrate, phosphorycholine and arabinoside. The main objective of this study was to determine new phenotypic characteristics that allowed a better characterization of Prototheca spp. Usage of recent bioinformatic tools improved the analyses of several features that are important for a better characterization of Prototheca spp.
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bovine mastitis associated with Prototheca blaschkeae
Journal of Clinical Microbiology, 2008Co-Authors: Sara Marques, Eliane Silva, J Carvalheira, Volker A R Huss, Christine Kraft, Arnaldo Videira, Gertrude ThompsonAbstract:Bovine mastitis is an important and complex disease responsible for economic losses in the dairy industry. Biotype II strains of the green alga Prototheca zopfii can be involved, most often resulting in chronic mastitis of difficult treatment associated with reduced milk production. This type of infection is rare, but the number of reported cases is increasing worldwide. In order to determine the kind of species involved in mastitis by Prototheca in northwest Portugal, 41 Prototheca isolates were genetically characterized. The algae are part of Prototheca isolates that were collected during a 6-year period, isolated from the milk of 41 dairy cows in a total of 22 herds with a history of increasing somatic cell counts, mild clinical signs of udder infection, and unsuccessful response to the usual therapy. PCR amplification of the 18S ribosomal DNA (rDNA), amplified rDNA restriction analysis, and phylogenetic analyses of the 18S rDNA sequences were performed. Thirty-seven isolates were identified as P. zopfii var. hydrocarbonea and four as Prototheca blaschkeae. These data suggest a high incidence of P. zopfii var. hydrocarbonea mastitis in the region and demonstrate for the first time the involvement of P. blaschkeae with bovine mammary gland infections.
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short communication in vitro antimicrobial susceptibility of Prototheca wickerhamii and Prototheca zopfii isolated from bovine mastitis
Journal of Dairy Science, 2006Co-Authors: Sara Marques, Eliane Silva, J Carvalheira, Gertrude ThompsonAbstract:Bovine mastitis caused by Prototheca spp. can assume high significance because of economic losses and the potential risk to public health. Studies on the susceptibility of Prototheca spp. to antimicrobials have demonstrated its high level of resistance. We report the susceptibility of bovine isolates of Prototheca wickerhamii and Prototheca zopfii to amphotericin B and nystatin, 2 antifungal agents commonly used in the control of protothecosis, and discuss the results. After subculture, minimum inhibitory concentrations of both antifungal drugs were determined using macrodilution and agar diffusion methods. The inoculum concentration was standardized by determination of colony-forming units per milliliter. Nystatin showed more efficacy than amphotericin B in inhibiting P. wickerhamii growth. In contrast, growth inhibition of P. zopfii was similar for both antifungal agents. This study demonstrates different in vitro susceptibility patterns of P. wickerhamii and P. zopfii, reinforcing the necessity for more investigation into drugs that can be used with clinical efficacy.
Pietro Buzzini - One of the best experts on this subject based on the ideXlab platform.
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a comparative study of the in vitro activity of iodopropynyl butylcarbamate and amphotericin b against Prototheca spp isolates from european dairy herds
Journal of Dairy Science, 2017Co-Authors: Tomasz Jagielski, Benedetta Turchetti, M Ricchi, Zofia Bakula, Henryk Krukowski, S Di Mauro, C Casciari, V Cambiotti, Elisabetta Manuali, Pietro BuzziniAbstract:ABSTRACT The objective of this study was to assess the in vitro effect of iodopropynyl butylcarbamate (IPBC) and amphotericin B (AMB) on Prototheca zopfii genotype 2 and Prototheca blaschkeae isolates recovered from dairy herds of Belgium, France, Italy, Germany, and Poland. The combination of IPBC with AMB on Prototheca isolates and toxicity of IPBC to the bovine mammary epithelial cells were also evaluated. The in vitro activity of IPBC and AMB against 96 isolates of P. zopfii genotype 2 and 42 isolates of P. blaschkeae was performed. Minimum inhibitory concentrations (MIC) and minimum algicidal concentrations (MAC) of IPBC and AMB were determined. To determine any synergistic, additive, or antagonistic effect of the combination of IPBC and AMB, 2-dimensional checkerboard combination tests were also performed to calculate fractional inhibitory concentrations. Cytotoxicity analysis of IPBC to the bovine mammary epithelial cell line was performed using a 3-(4,5-dimethyl-2-thiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. The MIC for 50 and 90% of isolates (MIC 50 and MIC 90 , respectively) for IPBC were 4 and 8 mg/L versus 0.5 and 1 mg/L for AMB, respectively. The MIC profiles differed between P. zopfii genotype 2 and P. blaschkeae , with the latter species being more susceptible to both compounds. The MIC 50 and MIC 90 of IPBC were 4 and 8 mg/L for P. zopfii genotype 2 and 1 and 2 mg/L for P. blaschkeae , respectively. The MIC 50 and MIC 90 of AMB were both 1 mg/L for P. zopfii genotype 2 and 0.25 and 1 mg/L for P. blaschkeae , respectively. Both IPBC and AMB exhibited the ability to kill Prototheca spp. The MAC for 90% of isolates of IPBC was twice the MIC 90 , whereas an 8-fold increase of the MIC 90 was algicidal in the case of AMB. Overall, the combined use of IPBC and AMB exhibited an increased algicidal effect, albeit the fractional inhibitory concentration index showed synergistic activity only against 3 P. zopfii genotype 2 isolates. For all the remaining isolates (87.5%), this combination produced only an additive effect. The MTT assay results showed both IPBC and AMB, at the concentrations employed in the study, to be nontoxic to the epithelial mammary gland cells (cell viability >90%). Notably, only IPBC at the highest concentration (i.e., 8 mg/L) exerted a slight cytotoxic effect on the cell line tested (mean cell viability: 88.54 ± 3.88 and 90.66 ± 3.0, after 2 and 4 h of MTT treatment, respectively). The anti- Prototheca activity of IPBC was here demonstrated for the first time. In addition, the combined use of IPBC with AMB enhanced each other's effect, creating an additive rather than synergistic interaction. Both agents, used at concentrations corresponding to MIC values against Prototheca spp., showed no toxic effect for the mammary epithelial cells. In conclusion, IPBC, used either alone or in combination with AMB, can be considered a promising option in the treatment armamentarium for Protothecal mastitis in dairy cows.
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first outbreak of bovine mastitis caused by Prototheca blaschkeae
Veterinary Microbiology, 2013Co-Authors: M Ricchi, G Cammi, N Arrigoni, Pietro Buzzini, C De Cicco, M Cammi, C GarbarinoAbstract:Abstract The most important animal disease caused by yeast-like algae belonging to the genus Prototheca is bovine mastitis. Although the infection can be caused by both Prototheca zopfii genotype 2 and Prototheca blaschkeae , the bulk of prevalence of bovine Protothecal mastitis has been so far attributed to the former, being P. blaschkeae only sporadically isolated. However, we report here the first outbreak of bovine mastitis caused by P. blaschkeae in an Italian dairy herd. One hundred and four individual milk samples, three bulk tank milk and 16 environmental samples within the herd were screened for the presence of Prototheca : five, one and four positive samples, were respectively observed. Molecular analysis revealed that, with the sole exception of one environmental isolate belonging to P. zopfii genotype 2, all Prototheca strains were identified as P. blaschkeae . Our results might suggest that even P. blaschkeae can induce mastitis outbreaks, while it is not clear if the higher incidence of P. zopfii genotype 2 as causative agent of Protothecal mastitis could reflect an intrinsic higher pathogenicity or it could be simply the consequence of its, so far observed, higher diffusion in worldwide dairy herd ecosystems.
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multicentre etest evaluation of in vitro activity of conventional antifungal drugs against european bovine mastitis Prototheca spp isolates
Journal of Antimicrobial Chemotherapy, 2012Co-Authors: Tomasz Jagielski, P E Lagneau, Eva Branda, Benedetta Turchetti, Pietro Buzzini, Uwe Roesler, H Lassa, Edward Malinowski, Angela Polleichtner, Sara MarquesAbstract:OBJECTIVES: Bovine mammary protothecosis is a serious pathology that entails high economic losses in the dairy industry. The disease, the frequency of which has recently been increasing worldwide, is caused by unicellular, achlorophyllous, yeast-like algae of two species: Prototheca zopfii and Prototheca blaschkeae. The objective of this study was to investigate the in vitro activity of a panel of conventional antifungal drugs against Prototheca spp. isolates. METHODS: A total of 144 P. zopfii genotype 2 and P. blaschkeae strains isolated from milk of mastitic cows were subjected to drug susceptibility testing by Etest methodology. RESULTS: Five out of ten antifungal drugs tested exhibited no activity against Prototheca spp. isolates. The best activity against Prototheca spp. was demonstrated by amphotericin B (MIC₉₀ of 1.5 mg/L). The MICs differed significantly (P < 0.01) between P. zopfii genotype 2 and P. blaschkeae, with the latter species being more susceptible to amphotericin B and azoles. Marked differences (P < 0.05) in azole and amphotericin B activities were noted among Prototheca spp. isolates originating from different European countries. Based on the correlation coefficients, a considerable cross-interaction was found among MICs of azoles and between MICs of azoles and amphotericin B for Prototheca spp. (P < 0.03). CONCLUSIONS: This study represents the largest, cross-European evaluation of antifungal activity against Prototheca spp. to date. The activity of amphotericin B against Prototheca spp. validates its potential use as a therapeutic agent against bovine protothecosis. For laboratory testing of drug activity against Prototheca spp., the Etest method is encouraged, due to its technical simplicity, rapidity and high intra- and inter-laboratory reproducibility.
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molecular characterization of Prototheca strains isolated from italian dairy herds
Journal of Dairy Science, 2010Co-Authors: M Ricchi, Marta Goretti, Eva Branda, G Cammi, C Garbarino, Benedetta Turchetti, Pablo Moroni, N Arrigoni, Pietro BuzziniAbstract:One hundred sixty-one Prototheca spp. strains isolated from composite milk and barn-surrounding environmental samples (bedding, feces, drinking, or washing water, surface swabs) of 24 Italian dairy herds were characterized by genotype-specific PCR analysis. Overall, 97.2% of strains isolated from composite milk samples were characterized as Prototheca zopfii genotype 2, confirming its role as the main mastitis pathogen, whereas Prototheca blaschkeae was only sporadically isolated (2.8%). Regarding environmental sampling, 84.9% of isolates belonged to P. zopfii genotype 2, 13.2% to P. blaschkeae, and 1.9% to P. zopfii genotype 1. The data herein contradict previous hypotheses about the supposed exclusive role of P. zopfii genotype 2 as the causative agent of Protothecal mastitis and, on the contrary, confirm the hypothesis that such pathology could be caused by P. blaschkeae in a few instances.
