Evaluation of the susceptibility of Prototheca zopfii to milk pasteurization

Protothecosis has been reported in humans (gastroenteritis, bursitis, etc.) and in many other animal species. Bovine mastitis represents the main form of occurrence of protothecosis in cattle. Milk as well as dairy products, when contaminated with Prototheca spp., represent a potential means of transmission of this zoonosis. The purpose of this study was to evaluate the susceptibility of forty Prototheca zopfii strains isolated from milk from intramammary infections in dairy cows and also from bulk milk tanks of dairy farms, to the different ratios of temperature/time employed in the thermal treatment of milk: 72–75 °C/1 5 seconds, 72–75 °C/20 seconds and 62–65 °C/30 minutes. The samples were subjected to these different temperature/time ratios. The evaluation of the thermal susceptibility of the P. zopfii strains showed that 34 strains were resistant in at least one of the tests. The results point out the need to consider the importance of mastitis caused by Prototheca spp. asrepresenting a public health risk. This revised version was published online in June 2006 with corrections to the Cover Date.     

First large-scale isolation of Prototheca zopfii from milk produced by dairy herds in Italy

A total of 1045 milk samples collected from infected and non infected quarters of 269 cows were investigated. This study showed that 4.7% of samples possessed cells of Prototheca spp. (10⁶cells/ml). The presence of other pathogenic microorganisms was also monitored. Prototheca spp. isolates were classified on the basis of current taxonomic guidelines and identified as P. zopfii. Susceptibility tests carried out in vitro by using 25 antibiotic compounds revealed that the strains of P. zopfii. were susceptible only to nystatin and amphotericin B (58 and 33% of total strains, respectively). The present study represents the first large-scale investigation carried out in Italy on the isolation of this achlorophyllous yeast-like microalga in milk samples produced by dairy herds.     

Epidemiologic study of environmental sources in a Prototheca zopfii outbreak of bovine mastitis

Bovine mastitis represents the main form of occurrence of protothecosis in animals. The detection of mastitis caused by Prototheca sp. indicates a serious problem which can affect an entire herd. The purpose of this study is to explain some aspects of the epidemiology of mastitis due to Prototheca zopfii with the evaluation of the presence of these microorganisms in samples collected from potential sources in the dairy herd. This study was performed during a Prototheca zopfii outbreak of clinical bovine mastitis in the State of São Paulo, Brazil. The following samples were aseptically collected for microbiological examination: milk (n = 211); rectal swabs (from 15 calves and 2 lactating cows); swabs from teat cup rubbers during milking (n = 2); water (n = 6); soil (n = 6). Prototheca zopfii was isolated from 77 (36.49%) of the 211 milk samples; 11 calves and 2 cows showed Prototheca zopfii in faecal samples; both swabs collected from the teat cup rubbers showed viable forms of Prototheca zopfii; this microorganism was also isolated from 2 water samples, and 1 soil sample collected from the dry cow pasture. Prototheca zopfii seemed to be widespread throughout the dairy herd environment where this outbreak of bovine mastitis occurred.This revised version was published online in October 2005 with corrections to the Cover Date.     

A novel DNA extraction and duplex polymerase chain reaction assay for the rapid detection of Prototheca zopfii genotype 2 in milk

Aims: To develop a PCR‐based assay to detect Prototheca zopfii (P. zopfii) and its mastitis‐related subtype (genotype 2) directly from milk samples. Methods and Results: The DNA extraction method herein is based on the lysing properties of chemical agents, mechanical grinding and DNA‐binding properties of silica particles; this method was developed to rapidly extract DNA directly from P. zopfii in bovine milk. Two pairs of primers specific for P. zopfii and genotype 2 were used in the duplex PCR, and a sensitivity test showed that the detection level was 5 × 10² colony‐forming units (CFU) ml^?1 for P. zopfii and 5 × 10³ CFU ml^?1 for genotype 2. Furthermore, a practical survey of 23 milk samples showed that the assay produced results that were in accordance with those obtained by the conventional microbiology method. Conclusions: The DNA extraction method is effective in isolating sufficient quantities of DNA from P. zopfii in milk for PCR analysis. The PCR assay is economical, sensitive and more rapid than the conventional culture method. Significance and Impact of the Study: The assay could be used as an alternative method for the rapid the detection of bovine mastitis resulting from P. zopfii genotype 2.     

Isolation of Prothoteca zopfii from a case of bovine mastitis in Brazil

The isolation of Prothoteca zopfii, an algae lacking chlorophyll, from bovine mastitic milk, is described herein. The isolation was performed on 8% sheep blood agar, following incubation at 37 °C for 48 h. Based on biochemical tests, susceptibility to clotrimazole, and light and electron microscopic observation of cellular morphology the algae was identified as P. zopfii . The affected animal did not improve following treatment and had to be eliminated. This revised version was published online in June 2006 with corrections to the Cover Date.     

Prototheca zopfii: Natural,transient, occurrence in pigs and rats

Domestic swine faeces and fresh faeces from trapped barnyard rats were heavily contaminated with Prototheca zopfii, a cause of dairy cow mastitis. When the pigs and rats were maintained on Prototheca-free diets, the transient intestinal population of P. zopfii decreased precipitously and disappeared. When combined with the information that other farm animals excrete P. zopfii, it was concluded that contaminated animal feed may be the source of large numbers of P. zopfii in the farm environment. We found P. zopfii in wet spoiled feed. Rats are logical vectors for contamination of feed.     

Activity of biogenic silver nanoparticles against isolates of Prototheca species from bovine mastitis

Prototheca spp. cause numerous infections in a wide variety of species, including treatment-unresponsive mastitis. Thus, the search for an effective therapy is essential. Silver nanoparticles are compounds with high therapeutic potential. This study aimed to evaluate the susceptibility profile and morphological changes in Prototheca spp. treated with biogenic silver nanoparticles (Bio-AgNP). The algaecide activity was evaluated in microplates by microdilution method, resulting in a MIC₅₀ of 30 μg ml⁻¹ and a MIC₉₀ of 60 μg ml⁻¹. Scanning electron microscopy demonstrated changes in the surface of Prototheca bovis cells following treatment. The algaecide activity of Bio-AgNP suggests a therapeutic potential as a novel approach for the control of Prototheca spp. in bovine mastitis.     

The purification and properties of malonic semialdehyde oxidative decarboxylase from Prototheca zopfii

1. An enzyme, which in the presence of NAD(+) and CoA oxidizes malonic semialdehyde to acetyl-CoA, has been purified from an extract of the colourless alga Prototheca zopfii. 2. The purified enzyme has optimum pH7.5, is specific for NAD(+) and requires a thiol compound for maximum activity. 3. The enzyme is inhibited by arsenite, N-ethylmaleimide and urea. 4. The results are discussed in relation to those obtained by other workers with a similar bacterial enzyme, and a possible reaction sequence is proposed.     

Effect of milk on antibacterial activity of tetracycline against Escherichia coli and Staphylococcus aureus isolated from bovine mastitis

The susceptibility of mastitis-causing Escherichia coli and Staphylococcus aureus to two commonly used antibiotics, tetracycline and penicillin G, was tested in raw milk and in Muller–Hinton (MH) broth by introducing a pH indicator, bromocresol purple, which was shown to be a simple, sensitive, and rapid method. The minimum inhibitory concentration (MIC) of penicillin G in milk was the same as those in MH broth, whereas the MIC of tetracycline in milk was 4 to 32 times that in MH. An irreversible binding between tetracycline and large molecules of milk, which might be due to a hydrophobic interaction, was demonstrated by a dialysis test, suggesting the observed impairing effect was due to the action of milk on the tetracycline being tested. Further investigation revealed that much of the reduction of tetracycline’s activity in milk was attributable to the milk protein casein, while other heat-sensitive components in milk also play some roles.     

Susceptibility and features of the ultrastructure of Prototheca zopfii following exposure to copper sulphate,silver nitrate and chlorexidine

One of the most important forms of the occurrence of protothecosis is bovine mastitis. Studies on the in vivo and in vitro susceptibility to antimicrobials have shown that the microorganism is resistant to most of them. Looking for alternative treatments this study aimed to study the susceptibility to copper sulphate (which has an important algicide effect) and silver nitrate (used in dairy cattle breeding for the cauterization of mammary glands) and also to chlorexidine (an important post-dipping anti-septic used in dairy practice), and the effect of these antimicrobials in the ultrastructure of Prototheca zopfii before and after the exposure to these drugs. The in vitro susceptibility tests to chlorexidine, silver nitrate and copper sulphate of the strains of Prototheca zopfii for the determination of their minimal microbicidal concentrations (MMC), were performed using the tube dilution method in Sabouraud dextrose broth and evaluation of colony growth after plating in Sabouraud dextrose agar. The MMCs of chlorexidine, copper sulphate and silver nitrate of the 50 strains tested were 0.01%, 0.1% and 0.3%, respectively. The tubes containing the material used in the antimicrobial susceptibility tests were prepared for the examination in an electron microscope. The untreated controls of P. zopfii showed a similar ultrastructural appearance with the typical characteristics of the microorganism. Cells exposed to silver nitrate showed changes suggesting thickness of the cell wall. Cells exposed to chlorexidine showed changes suggesting degradation of intra-cellular organelles present in the cytoplasm. P. zopfii treated with copper sulphate showed changes suggesting fibrilation of inner layer of cell wall.This revised version was published online in October 2005 with corrections to the Cover Date.     

Effects of some factors on protoplast formation of a microalga,Prototheca zopfii

The effects of experimental factors on protoplast formation of Prototheca zopfii Kru¨ger in 0.85 m NaCl using Macerozyme R-200 were studied based on a fractional factorial experimental design. The rate of protoplast formation was mainly affected by the incubation temperature and the age of algal cells. The optimal condition for the maximum protoplast yield was determined based on a response surface model. These were: mid-logarithmic phase cells and Macerozyme concentration of 4% at a temperature of 35°C.     

Two‐dimensional proteome reference map of Prototheca zopfii revealed reduced metabolism and enhanced signal transduction as adaptation to an infectious life style

Biochemical, serological, and genetic analyses have identified two genotypes of Prototheca zopfii, a unicellular microalga belonging to the family Chlorellaceae. The P. zopfii genotype 1, abundantly present in cow barns and environment, remains nonpathogenic, while P. zopfii genotype 2 has been isolated from cows with bovine mastitis. The present study was carried out to identify the protein expression level difference between the pathogenic and nonpathogenic strains of P. zopfii. A total of 782 protein spots were observed on the 2D fluorescence difference gel electrophoresis (2D DIGE) gels among which 63 and 44 proteins were identified to be overexpressed in genotypes 1 and 2, respectively. The limited number of protein entries specific for Prototheca in public repositories resulted mainly in the identification of proteins described in other algae, microorganisms, or plants. Gene ontology (GO) analysis indicated reduced carbohydrate metabolism in genotype 1, while genotype 2 displayed enhanced DNA binding, kinase activity, and signal transduction. These effects point to metabolic and signaling adaptations in the pathogenic strain and provide insights into the evolution of otherwise highly similar strains. All MS data have been deposited in the ProteomeXchange with identifier PXD000126.     

Expression of bovine lactoferrin and lactoferrin N-lobe by recombinant baculovirus and its antimicrobial activity against Prototheca zopfii.

Lactoferrin (LF) is a multifunctional, iron-binding glycoprotein found in secretory fluids of mammals. In this study, DNA encoding bovine lactoferrin (bLF) or the N-terminal half of bLF (bLF N-lobe) was inserted into a baculovirus transfer vector, and a recombinant virus expressing bLF or bLF N-lobe was isolated. An 80-kDa bLF-related protein expressed by the recombinant baculovirus was detected by monoclonal antibodies against bLF N-lobe and the C-terminal half of bLF (bLF C-lobe). A 43-kDa bLF N-lobe-related protein expressed by the recombinant baculovirus was detected by anti-bLF N-lobe monoclonal antibody, but not by anti-bLF C-lobe monoclonal antibody. These proteins were also secreted into the supernatant of insect cell cultures. Recombinant bLF (rbLF) and bLF N-lobe (rbLF N-lobe) were affected by tunicamycin treatment, indicating that rbLF and rbLF N-lobe contain an N-linked glycosylation site. Antimicrobial activity of these recombinant proteins against Prototheca zopfii (a yeast-like fungus that causes bovine mastitis) was evaluated by measuring the optical density of the culture microplate. Prototheca zopfii was sensitive to rbLF and rbLF N-lobe, as well as native bLF. There was no difference in antimicrobial activity between rbLF N-lobe and bLF C-lobe.     

Inhibition of bacteriophage K proliferation on Staphylococcus aureus in raw bovine milk

AIMS: To assess the ability of staphylococcal bacteriophage K to inhibit Staphylococcus aureus in raw milk. METHODS AND RESULTS: The ability of bacteriophage (phage) to replicate in milk is important in situations where phage might be used as a therapeutic for bovine mastitis. Phage K was able to replicate normally, leading to elimination of the host culture in milk, which had been previously heat-treated. When raw milk was used under identical conditions, the phages were unable to replicate. Phage adsorption assays were performed and these demonstrated that adsorption of phage was significantly reduced in the raw milk while it was restored in the heat-treated sample (86.50% compared with 99.96% adsorption respectively). When confocal microscopy with a Live/Dead Bac light staining system was employed, it was observed that in raw milk S. aureus formed clusters associated with fat globules, while in heat-treated milk, bacterial agglutination had not occurred. CONCLUSIONS: Raw milk inhibits staphylococcal phage K proliferation. Significance and Impact of the Study: This observation has implications for the exploitation of staphylococcal therapeutic phage in milk.     

Influence of a 3‐month training program on muscular damage and neutrophil function in male university freshman judoists

We studied the effects of a high intensity and high frequency 3‐month training program on muscle damage and neutrophil function in male judoists. The study included 15 male judoists who started intensive judo training program after a 6‐month break. Creatine kinase (CK), neutrophil counts and reactive oxygen species (ROS) production capability as well as phagocytic activity (PA) of neutrophils were measured at 2 stages; entering university (pre‐training) and after 3‐month training (post‐training). At both points, we investigated parameters three times: just before, immediately after and 24 h after a 2‐h practice session. Practice‐mediated change in CK was lower at post‐training than at pre‐training. Neutrophil count significantly increased after 2‐h practice but recovered 24 h later whereas it showed no subsequent and further increased at 24 h post‐practice. Although neutrophil ROS production capability and PA both decreased (breakdown) after practice session, ROS production capability increased and PA decreased (well‐adapted) at the post‐training. Long‐term training strengthened muscular function and improved neutrophil reaction against practice‐mediated stress. Copyright © 2012 John Wiley & Sons, Ltd.     

Further Observations on the Inhibition of Prototheca zopfii Growth by 3-Amino-l,2,4-triazole

The concentration of amitrole (3-amino-l,2,4-triazole) causing 50 per cent inhibition of Prototheca zopfii growth is 1.25 mg/100 g at 25°C. This inhibition is not reversed by adenine. 50 per cent inhibition of growth is also caused by 100–120 mg/ 100 g 2-aminopurine and this inhibition is partially reversed by 40 mg/100 g adenine, the highest adenine concentration tested due to solubility problems. Imidazoleglycerol accumulation occurs in the medium of cells grown in the presence of amitrole.