Molecular characterization of Polish Prototheca zopfii mastitis isolates and first isolation of Prototheca blaschkeae in Poland

Jagielski, Tomasz; Lassa, H.; Ahrholdt, Jennifer; Roesler, Uwe; Malinowski, E.

doi:10.2478/v10181-010-0025-0

Cited by 12 publications

(10 citation statements)

References 20 publications

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“…It is in agreement with the reports of Moller et al in Germany (Moller et al, 2007), Aouay et al in Belgium (Aouay et al, 2008), Osumi et al in Japan (Osumi et al, 2008), Jagielski et al in Poland (Jagielski et al, 2010), and Shahid et al in China (Shahid et al, 2016). We determined Bax protein expression, by western blotting, to further validate the apoptosis in bMECs infected with P. zopfii genotypes.…”

Section: Discussionsupporting

confidence: 92%

“…The invasion and survival of P. zopfii type-II in bMECs and its role in apoptosis is not comprehensively studied yet. Previous published studies regarding protothecal mastitis caused by P. zopfii are mostly focused on prevalence, molecular characterization of P. zopfii (Jagielski et al, 2010; Gao et al, 2012; Chang et al, 2013; Bozzo et al, 2014; Shahid et al, 2016), and on the determination of some immunogenic proteins and virulence determinants of P. zopfii genotype-II (Irrgang et al, 2015). Therefore, the current study was designed with the objective to evaluate the ultrastructural features of apoptosis and to study the comparative apoptotic potentials of P. zopfii genotype-I and -II on bMECs.…”

Section: Introductionmentioning

confidence: 99%

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Prototheca zopfii Induced Ultrastructural Features Associated with Apoptosis in Bovine Mammary Epithelial Cells

Shahid

Wang

et al. 2017

Front. Cell. Infect. Microbiol.

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Prototheca zopfii infections are becoming global concerns in humans and animals. Bovine protothecal mastitis is characterized by deteriorating milk quality and quantity, thus imparting huge economic losses to dairy industry. Previous published studies mostly focused on the prevalence and characterization of P. zopfii from mastitis. However, the ultrastructural pathomorphological changes associated with apoptosis in bovine mammary epithelial cells (bMECs) are not studied yet. Therefore, in this study we aimed to evaluate the in vitro comparative apoptotic potential of P. zopfii genotype-I and -II on bMECs using flow cytometry, scanning electron microscopy (SEM), and transmission electron microscopy (TEM). The results showed fast growth rate and higher adhesion capability of genotype-II in bMECs as compared with genotype-I. The viability of bMECs infected with P. zopfii genotype-II was significantly decreased after 12 h (p < 0.05) and 24 h (p < 0.01) in comparison with control cells. Contrary, genotype-I couldn't show any significant effects on cell viability. Moreover, after infection of bMECs with genotype-II, the apoptosis increased significantly at 12 h (p < 0.05) and 24 h (p < 0.01) as compared with control group. Genotype-I couldn't display any significant effects on cell apoptosis. The host specificity of P. zopfii was also tested in mouse osteoblast cells, and the results suggest that genotype-I and -II could not cause any significant apoptosis in these cell lines. SEM interpreted the pathomorphological alterations in bMECs after infection. Adhesion of P. zopfii with cells and further disruption of cytomembrane validated the apoptosis caused by genotype-II under SEM. While genotype-1 couldn't cause any significant apoptosis in bMECs. Furthermore, genotype-II induced apoptotic manifested specific ultrastructure features, like cytoplasmic cavitation, swollen mitochondria, pyknosis, cytomembrane disruption, and appearance of apoptotic bodies under TEM. The findings of the current study revealed that genotype-II has the capability to invade and survive within the bMECs, thus imparting significant damages to the mammary cells which result in apoptosis. This study represents the first insights into the pathomorphological and ultrastructure features of apoptosis in bMECs induced by P. zopfii genotype-II.

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Section: Discussionsupporting

confidence: 92%

Section: Introductionmentioning

confidence: 99%

Prototheca zopfii Induced Ultrastructural Features Associated with Apoptosis in Bovine Mammary Epithelial Cells

Shahid

Wang

et al. 2017

Front. Cell. Infect. Microbiol.

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“…Primary isolation and culturing of algal isolates were performed according to standardized procedures, essentially described elsewhere (Jagielski et al, 2010). Speciation was based on micromorphological characterization of lactophenol-cotton-blue-stained culture smears and carbohydrate assimilation profiling with the API 20C AUX system (bioMérieux, Marcy l'Etoile, France).…”

Section: Prototheca Spp Isolates and Bovine Mammary Epithelial Cellsmentioning

confidence: 99%

A comparative study of the in vitro activity of iodopropynyl butylcarbamate and amphotericin B against Prototheca spp. isolates from European dairy herds

Jagielski

Bakuła

Mauro

et al. 2017

Journal of Dairy Science

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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 and MIC, 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 and MIC 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 and MIC 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, whereas an 8-fold increase of the MIC 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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“…Genotype 2 was assigned to be the most prevalent responsible for bovine protothecal mastitis, whereas genotype 1 was considered to be not pathogenic (Roesler et al, 2006;Möller et al, 2007). Prototheca blaschkeae was primarily isolated from a human onychomycosis (Roesler et al, 2006), and then for the first time from bovine mastitis in Portugal (Marques et al, 2008(Marques et al, , 2010Thompson et al, 2009); meanwhile, P. blaschkeae incidences have been reported globally (Jagielski et al, 2010;Gao et al, 2012;Sobukawa et al, 2012;Ricchi et al, 2013).…”

Section: Introductionmentioning

confidence: 99%

Internal transcribed spacer sequence-based rapid molecular identification of Prototheca zopfii and Prototheca blaschkeae directly from milk of infected cows

Marques

Huss

Pfisterer

et al. 2015

Journal of Dairy Science

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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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Molecular characterization of Polish Prototheca zopfii mastitis isolates and first isolation of Prototheca blaschkeae in Poland

Cited by 12 publications

References 20 publications

Prototheca zopfii Induced Ultrastructural Features Associated with Apoptosis in Bovine Mammary Epithelial Cells

Prototheca zopfii Induced Ultrastructural Features Associated with Apoptosis in Bovine Mammary Epithelial Cells

A comparative study of the in vitro activity of iodopropynyl butylcarbamate and amphotericin B against Prototheca spp. isolates from European dairy herds

Internal transcribed spacer sequence-based rapid molecular identification of Prototheca zopfii and Prototheca blaschkeae directly from milk of infected cows

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