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.     

Characterization of <Emphasis Type="Italic">Prototheca zopfii</Emphasis> Associated with Outbreak of Bovine Clinical Mastitis in Herd of Beijing,China

Prototheca zopfii (P. zopfii) has become an important cause of bovine mastitis in many countries. In the present study, to better understand the occurrence of one clinical mastitis (CM) outbreak due to P. zopfii, the molecular characterization and resistance patterns of the microalga were described. P. zopfii strains were isolated from 17 of 23 quarters, which suffered CM in the outbreak, and 7 of 46 CM recovered quarters before the outbreak, as well as 2 of 75 environmental samples in the dairy farm. All strains were identified as genotype 2 by genotype-specific PCR analysis. Results of in vitro antimicrobial and antifungal susceptibility tests indicated that these strains were resistant to majority of tested drugs, with the only exception of amphotericin B, nystatin, streptomycin, gentamicin, and amikacin. This is the first report about CM outbreak caused by P. zopfii in China. These data suggest that P. zopfii may represent a serious risk in the studied herd, and this microalga could be an important potential pathogen causing mastitis in dairy herds of Beijing.     

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.     

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.     

Evaluation of the efficacy of chemical disinfectants for disinfection of heat‐polymerised acrylic resin

doi: 10.1111/j.1741‐2358.2010.00400.x
Evaluation of the efficacy of chemical disinfectants for disinfection of heat‐polymerised acrylic resin Objective: This study evaluated the efficacy of disinfectants on the internal aspect of heat‐polymerised acrylic resin contaminated with microbial strains. Background: Dentures absorb oral fluids and become contaminated by different microorganisms. Methods: Two hundred and fifty rectangular specimens were made of heat‐polymerised acrylic resin, and then divided into five groups corresponding to the microbial strains (Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans, S. mutans and Enterococcus faecalis). After contamination, the specimens were immersed in 1 and 2% sodium hypochlorite and 2% glutaraldehyde for periods of 5, 10 and 15 min. The specimens were placed into tubes containing different broths and incubated at 35°C and then visually analysed. Turbidity in the medium indicated microbial growth. The Fisher’s exact test was used in the analysis of the results. Results: The strain E. faecalis was the most resistant to the disinfectant solutions, and among them, glutaraldehyde was more effective than 2 and 1% hypochlorite for disinfection for 5 min; in the 10‐min period there were no differences between the disinfectants. In 15 min of immersion, 1% hypochlorite and glutaraldehyde were more effective than 2% hypochlorite. Conclusions: Disinfection for 10 min with 1% hypochlorite and glutaraldehyde is effective in disinfecting the internal aspect of heat‐polymerised acrylic resin.     

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.     

A rapid real-time PCR/DNA resolution melting method to identify Prototheca species

Aim: The study describes the development of a simple and rapid tool to identify yeast‐like microalgae belonging to the genus Prototheca. Methods and Results: The method, based on two‐step Real Time PCR reaction followed by DNA Resolution Melting Analysis (qPCR/RMA), has been developed using reference strains belonging to both pathogenic (P. zopfii genotype 2, P. wickerhamii and P. blaschkeae) and non‐pathogenic species (P. zopfii genotype 1, P. stagnora and P. ulmea). In order to validate the method, seventy recently isolated Prototheca strains were thus tested in parallel with both the first qPCR/RMA and the conventional genotype‐specific PCR assay: they were classified as P. zopfii genotype 1, P. zopfii genotype 2 and P. blaschkeae, with a perfect accordance between the two above methodologies. Furthermore, we used the second qPCR/RMA to identify the other species (P. stagnora, P. ulmea and P. wickerhamii), which cannot be discriminated by conventional PCR assay. Conclusions: The assay two‐step Real Time PCR is accurate, robust, cost‐effective and faster than auxonographical, biochemical or conventional molecular biology methods. Significance and Impact of the Study: the rapid and high throughout two‐step qPCR/RMA tool can be usefully used for the identification of clinical and environmental Prototheca species into the framework of the diagnosis of animal (e.g. bovine mastitis) or human protothecosis.     

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.     

Prevalence of Prototheca spp. on dairy farms in Poland – a cross-country study

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.     

Treatment with Gentamicin on a Murine Model of Protothecal Mastitis

The aim of this study was to establish a murine protothecal mastitis model and to evaluate the treatment efficiency of gentamicin. Challenge routes were determined with a pathogenic Prototheca zopfii genotype 2 (P. zopfii) strain. 25 BALB/c mice were inoculated in mammary glands with graded dosages (10³, 10⁴, 10⁵, 10⁶, 10⁷ CFU of P. zopfii) and killed on the 7th day. Another 25 animals were also killed at 1, 3, 5, 7, and 9 days after inoculation of 1 × 10⁶ CFU of P. zopfii, the milk somatic cell counts, pathological section of mammary glands, and P. zopfii burden were observed. The antimicrobial activity was tested using disc diffusion test and minimum inhibitory concentrations. Gentamicin was given intramuscularly to analyze the therapeutic effect. The results showed that the best infection route was intra-mammary gland, and the mastitis model was established with 1 × 10⁶ CFU of P. zopfii. After infection, the somatic cell counts increased significantly. The pathological reaction mainly consisted of infiltration of inflammatory cells, destruction of acini, accumulation of lymphocyte cells and the severity of the changes was dosage and time-dependent. The P. zopfii burden revealed that P. zopfii continuously replicated. In vitro susceptibility tests indicated that the Prototheca strains were antimicrobial susceptible to gentamicin at concentrations between 0.03 and 4 μg/ml. In vivo therapeutic assay demonstrated that high concentrations of gentamicin (≥20 mg/kg) could inhibit the growth of P. zopfii. We conclude that the murine model of protothecal mastitis was established successfully and gentamicin may be an effective choice for treatment of P. zopfii.     

常用消毒剂对布鲁氏菌疫苗株的灭菌效果评估

[背景]布鲁氏菌是一种重要的人畜共患胞内寄生菌,是引起动物和人发生布鲁氏菌病(布病)的病原体.高效消毒剂杀灭环境中的布鲁氏菌是防控布病的关键措施.[目的]评价市面上常用消毒剂对布鲁氏菌的灭菌效果及差异.[方法]选用市面上9种不同类型的常用消毒剂,以高、中、低3种不同使用浓度分别研究其对牛种、羊种和猪种布鲁氏菌的杀灭效果...     

Efficacy of some commerical disinfectants against the bacterial isolates from a poultry slaughterhouse in Turkey

In this study, efficacy of seven different commercial disinfectant preparations was investigated against characteristic bacteria of a poultry slaughterhouse in Ankara (Turkey) by using paper disc-agar diffusion method and surface effectiveness test. According to paper disc-agar diffusion method, some disinfectants had wide efficacy against the test bacteria. The disinfectant effectiveness generally increased by the increasing of disinfectant concentration. However, some disinfectants were ineffective even though at their highest concentrations. The most effective disinfectant was B which contains QAC as active agent.Staphylococcus spp.,Staphyloccus aureus andEscherichia coli were the most sensitive bacteria to the disinfectants. Some pathogenic isolates, especiallySalmonella spp. andCampylobacter spp., were the most resistant ones to many of the disinfectants tested. The results of paper disc-agar diffusion method indicated the importance of characteristic bacterial strains of food plants as the test bacteria for disinfectant efficacy tests. Conversely of paper disc-agar diffusion method, all disinfectants were effective against the isolates by surface effectiveness test depending on exposure time. The disinfectants, except A and F, produced at least 3 log unit reduction during 5 min of exposure. However, all disinfectants at their lowest concentrations were effective against all tested bacteria during 15 and 30 min by this test.     

Effect of Prototheca zopfii on neutrophil function from bovine milk

This study was carried to investigate neutrophil function in the presence of Prototheca zopfii. For this purpose, bovine milk neutrophils were incubated in the absence (control) of and presence of P. zopfii, and then they were examined hydrogen peroxide (H₂O₂) production, antioxidant enzyme activities, and phagocytic capacity. Milk was collected from negative “California Mastitis Test” (CMT) quarter from three lactating Holstein cows after induction of leukocytosis with an intramammary infusion of oyster glycogen. H₂O₂ production was measured using the phenol red method. Catalase activity was measured following H₂O₂ reduction at 240 nm and the activity of glutathione reductase was determined by measuring the rate of NADPH oxidation at 340 nm. P. zopfii death was assessed by fluorescent microscopy using acridine orange assay and by colony forming units (CFUs). Comparisons between the groups were initially performed by analysis of variance (ANOVA). Significant differences were then compared using Tukey’s test with a significance coefficient of 0.05. Hydrogen peroxide production, catalase and glutathione reductase activities by neutrophils incubated in presence of P. zopfii were stimulated five times, 21% and 27% respectively, compared to the unstimulated-neutrophils. Neutrophils did not affect P. zopfii death as shown by microscopy and CFUs. These observations led to the conclusion that the P. zopfii promote a high increase of H₂O₂ production by neutrophils from bovine milk during algae exposition accompanied by increase of antioxidant enzyme activities; however, this process did not affect P. zopfii death.     

In Vitro Susceptibility of Prototheca to pH and Salt Concentration

Prototheca sp. can assume high economic significance in the dairy industry and pose a potential risk for the public health. We investigated the in vitro susceptibility of Prototheca isolates retrieved from mastitic milk (P. zopfii and P. blaschkeae) to different pH buffers and salt concentrations using a microbroth assay adapted from the Clinical Laboratory Standards Institute guidelines. Different pH buffer solutions ranging from pH 1 to pH 12 and different sodium chloride concentrations, 4.5, 9 and 18%, were tested. P. zopfii strains presented an optimal growth between pH 5 and 9, a complete growth inhibition at pH 3, and limited growth at pH 1 and 12, whereas P. blaschkeae strains showed higher susceptibility to all pH values except for pH 3 where it demonstrated a moderate growth when compared to P. zopfii strains. When salinity was incremented, P. blaschkeae was more resistant than P. zopfii, although a reduction in growth for all strains of Prototheca was observed. This study demonstrated differences in the in vitro susceptibilities of P. zopfii and P. blaschkeae to different pH and salt concentrations and intend to be a contribution on the understanding of some of the physiologic features that can be associated with the survival of these microalgae in the environment.     

Effectiveness of selected premilking teat-cleaning regimes in reducing teat microbial load on commercial dairy farms

Aims: To determine the effectiveness of premilking teat‐cleaning regimes in reducing the teat microbial load and effect on milk quality. Methods and Results: The effectiveness of several premilking teat‐cleaning regimes in reducing teat microbial load was assessed using 40 cows on each of the four commercial UK dairy farms with herringbone parlours during two sampling periods. In the first experiment, all the treatments reduced teat total viable count (TVC), but there was no significant difference between the hypochlorite wash and dry wipe, iodine dip and dry and alcohol‐medicated wipe or dry wipe alone. In the second experiment, the chlorine wash and dry wipe was significantly more effective in reducing teat TVC than a water wash and dry, chlorine dip and dry or a dry wipe. There was no relationship between cleaning regime and milk TVC, Enterobacteriaceae or Escherichia coli levels. Conclusions: All of the cleaning techniques studied reduced teat microbial load, however, the chlorine wash and dry was the most effective. Significance and Impact of the study: The premilking teat‐cleaning techniques studied reduced the teat microbial load and therefore reduced the potential for milk contamination; however, a wash including an effective disinfectant followed by a dry wipe was the most effective.     

Impact of prescribed cleaning and disinfectant use on microbial contamination in the home

Aims: To identify and quantify the presence of Escherichia coli, Staphylococcus aureus, Salmonella, hepatitis A and norovirus in households and to assess the effect of chlorine and quaternary ammonium–based disinfectants following a prescribed use. Methods and Results: Eleven sites distributed in kitchen, bathroom, pet and children′s areas of two groups of 30 homes each: (i) a nonprescribed disinfectant user group and (ii) a disinfectant protocol user group. During the 6‐week study, samples were collected once a week except for week one when sample collection occurred immediately before and after disinfectant application to evaluate the disinfectant protocol. The concentration and occurrence of bacteria were less in the households with prescribed use of disinfectants. The greatest reductions were for E. coli (99%) and Staph. aureus (99·9999%), respectively. Only two samples were positive for HAV, while norovirus was absent. Disinfection protocols resulted in a significant (P < 0·05) microbial reduction in all areas of the homes tested compared to homes not using a prescribed protocol. Conclusions: The study suggests that disinfectant product application under specific protocol is necessary to achieve greater microbial reductions. Significance and Impact of the Study: Prescribed protocols constitute an important tool to reduce the occurrence of potential disease‐causing micro‐organisms in households.     

Genotyping by sequencing reveals a new locus for pig teat number

Teat number is an extremely valuable trait for sow reproduction performance and piglet survival. Here, we used genotyping‐by‐sequencing and a general liner model to carry out genome‐wide analysis of sow total teat number in an Erhualian population. The results indicated that eight SNPs on chromosomes 4, 5, 9 and 10 were significant genome wide (Bonferroni method, P < 2.85E‐7) for sow total teat number. Validation analyses were performed in 298 Erhualian and 904 Large White sows using these significant SNPs and the general liner model procedure in sas . Finally, only the SNP on chromosome 5 was found to be significantly associated with sow teat number in both populations. The GG genotype individuals had 2.23 and 0.82 more teat numbers respectively than did the AA genotype individuals in these two populations (P < 0.05). According to the expression and annotation analyses, we inferred the presence of a gene or lincRNA that could affect teat number by regulating other genes and ultimately affecting the mammogenesis of pigs. Further studies using methods such as Cas9 editing and gene silencing analysis are necessary for additional function analysis.     

Removal of Viable Airborne Fungi from Indoor Environments by Benzalkonium Chloride-Based Aerosol Disinfectants

Fungi are ubiquitous in indoor environments, and some taxa can cause clinical symptoms in humans. Thus, from the viewpoint of public health, methods to reduce indoor airborne fungi are needed. The goal of this study was to examine the efficacies of benzalkonium chloride (BAC)–based aerosol disinfectants to remove airborne viable fungi from indoor environments. The laboratory- and field-based experiments were conducted to compare airborne culturable fungal concentrations before and after the disinfectant aerosol applications. The laboratory-based experiments showed the greater efficacies by the BAC-based disinfectant aerosol than by pure-water control aerosol (p <.05, t-test). In the field study using the BAC-based disinfectant aerosols, on average a 58% reduction of total airborne culturable fungal concentrations were observed. Additionally, the significant reduction was found for a group of airborne yeasts or yeast-like organisms (p <.05, Wilcoxon signed rank test). The BAC-based aerosol disinfectants are effective when used to reduce the numbers of airborne culturable fungi, in particular yeasts or yeast-like organisms, from indoor environments.     

Comparative proteomics lends insight into genotype‐specific pathogenicity

Comparative proteomic analyses have emerged as a powerful tool for the identification of unique biomarkers and mechanisms of pathogenesis. In this issue of Proteomics, Murugaiyan et al. utilize difference gel electrophoresis (DIGE) to examine differential protein expression between nonpathogenic and pathogenic genotypes of Prototheca zopfii, a causative agent in bovine enteritis and mastitis. Their findings provide insights into molecular mechanisms of infection and evolutionary adaptation of pathogenic genotypes, demonstrating the power of comparative proteomic analyses.     

Presence of qacEΔ1 gene and susceptibility to a hospital biocide in clinical isolates of Pseudomonas aeruginosa resistant to antibiotics

Biocides play an important role in healthcare-associated infection control by either minimizing or preventing microorganism dissemination. This study evaluated the susceptibility of Pseudomonas aeruginosa clinical isolates to a quaternary ammonium (QAC) disinfectant and antibiotics, and verified the presence of qacEΔ1, a determinant of resistance to QAC. The disinfectant test was the Association of Official Analytical Chemists Use-Dilution Test, and polymerase chain reaction was used to examine for qacEΔ1. The qacEΔ1 gene was detected in 48% of the isolates. Eighty-eight percent of the multiresistant isolates carried qacEΔ1 gene, while 35% of the non-multiresistant isolates was positive to this gene, and multiresistance well correlated with its presence. Among isolates tested for the disinfectant, 46% showed a reduced susceptibility to the disinfectant. qacEΔ1 gene was present in 70% of the susceptible isolates to the biocide, whereas 90% of the less susceptible strains harbored this gene. Reduced susceptibility to the disinfectant was independent of presence of qacEΔ1 suggesting that it does not play an important role in biocide resistance in P. aeruginosa. As far as we know, it is the first report confirming this fact and testing with disinfectant at its in-use concentration. The evidence of less susceptible strains than the reference bacterium used in disinfectant testing, and the high percentage of qacEΔ1 gene detected are of special concern and suggests continued investigation in laboratory and in situ, not only in healthcare settings, but also in all areas of biocide usage, including different micro-organisms and biocides.