Occurrence of Listeria monocytogenes in fresh and processed foods in Navarra (Spain)

The presence of Listeria spp. was investigated in a total of 3685 food samples obtained from different industries and markets of Northern Spain in the last 4 years. The samples analyzed include fresh raw products (meat, milk and poultry) and treated products (cooked and cured meats, frozen vegetables and smoked salmon). Occurrence of Listeria spp. varied from 8.1% in soft cheese to 76.3% in raw poultry samples. The highest incidence of L. monocytogenes also occurred in raw poultry (36.1% positive samples). Despite this high incidence of contamination, these kinds of products carry a low risk of listeriosis transmission because of the heat treatment prior to consumption. On the other hand, the ready-to-eat products (RTE) tested in this study showed incidences that could pose serious health problems, taking into account that the storage conditions may allow for rapid growth of the pathogen. It was also found that up to 75.5% of the L. monocytogenes strains isolated in this study belonged to serogroup 1, mainly serotype 1/2a, while the clinical cases observed in Navarra in the same period of time belonged mainly to serotype 4b/4bx.     

Occurrence of Campylobacter spp. in poultry and poultry products for sale on the Polish retail market

In 2007 and 2008, a monitoring study was carried out in Poland to examine the occurrence of thermotolerant Campylobacter spp. in raw and cooked chicken products available on the retail market. A total of 912 samples were tested: 443 samples of raw chicken meat, 146 samples of giblets, and 323 ready-to-eat poultry products (150 samples of spit-roasted chicken, 56 samples of smoked chicken, and 117 samples of paté and cold meats). A high level of contamination of raw chicken meat (51.7% of samples) and chicken giblets (47.3% of samples) was detected. However, thermotolerant Campylobacter spp. were found in only 1.2% of the ready-to-eat poultry products.     

Risk profiles of pork and poultry meat and risk ratings of various pathogen/product combinations

Risk profiles of pork and poultry meat were carried out using an Excel-based software program, Risk Ranger. It is a semi-quantitative risk estimator answering various questions relating to the probability of exposure to a hazard, susceptibility of the population of interest, severity of the illness caused by the hazard if present and probability of food containing an infectious dose. Therefore, qualitative and quantitative inputs were used to estimate and rank the risk of various hazards/food combinations. Risk scores provided by the tool were characterized as low, medium and high. Also, health risk was estimated separately, where needed, for low and high risk populations. Low risk scores were obtained for Salmonella spp., Listeria monocytogenes and enterohaemorrhagic Escherichia coli (EHEC) for low risk population. High risk scores were obtained for hepatitis E virus (HEV) in raw pork products (both low and high risk populations). Moderate risk scores for Salmonella spp. and L. monocytogenes in processed pork or poultry-meat products (ready-to-eat or to be reheated) and partially cooked pork products were also obtained (low risk population). Scores for Staphylococcus aureus, Clostridium perfringens and Bacillus cereus and various product types were mostly in the "medium" risk category, except for S. aureus/ready-to-eat pork products able to support growth of the organism, which fell into the high risk category. Campylobacter spp. gave moderate risk scores with one exception (raw poultry products), whereas Y. enterocolitica showed combinations of low risk and few of medium risk. High risk pathogen/product combinations identified were: 1) temperature abused, ready-to-eat pork and/or poultry-meat products with extended shelf life and cross-contaminated by L. monocytogenes (high risk population), EHEC (high risk population) or S. aureus (all population), 2) partially cooked or processed intended to be reheated pork products cross-contaminated by L. monocytogenes, served undercooked and receiving improper cooling or reheating (high risk population), and 3) all people consuming undercooked meals cross-contaminated with Campylobacter spp. (e.g. from raw poultry and raw poultry-meat products) and HEV (e.g. from raw pork and raw pork-meat products). Salmonellae gave high risk scores in all food categories (except preserved meat products) for high risk population. Preserved meats (mainly pork) such as dry fermented sausages gave low risk scores. Only Salmonella spp., L. monocytogenes and E. coli EHEC gave moderate risk ratings in case of ingredients likely to be contaminated at an early stage of processing (e.g. animal at slaughter) and inadequate fermentation process. These results may constitute a source of information for hazard assessment during application of a Food Safety Management System.     

9274份肉及肉制品食源性致病菌监测结果分析

目的 了解吉林省9274份肉及肉制品食源性致病菌污染情况,为防控食源性疾病提供科学依据。方法从吉林省9个地市级行政区采集市售6类肉及肉制品样品共9274份,包括生畜肉、生禽肉、熟肉制品、调理肉制品、冷冻肉糜制品和动物血液及制品。按照国家标准方法检测10种食源性致病菌。结果 全部9274份样本食源性致病菌总阳性检出率为3.9%(366/9274)。检出率最高为调理肉制品 (13.0%,63/483),其次是生禽肉(5.6%,107/1900)和生畜肉(5.0%,71/1428)。检出的主要致病菌为单核细胞增生李斯特菌、金黄色葡萄球菌和沙门菌。生禽肉中弯曲菌检出率(7.5%,31/411)和产气荚膜梭菌检出率(3.9%,7/180)均高于沙门菌检出率(3.5%,8/231)。生禽肉、生畜肉中未检出小肠结肠炎耶尔森菌。动物血液及制品未检出单细胞增生李斯特菌、弯曲菌和小肠结肠炎耶尔森菌。冷冻肉糜制品未检出沙门菌。熟肉制品未检出大肠埃希氏菌O157、志贺菌和蜡样芽胞杆菌。熟肉制品各年度检出率范围为1.3%-4.4%。结论 吉林省市售的肉及肉制品较长时间受到不同程度的食源性致病菌污染,存在食源性疾病发生的风险。     

Kitchen practices used in handling broiler chickens and survival of Campylobacter spp. on cutting surfaces in Kampala, Uganda

Cross-contamination during food preparation has been identified as an important factor associated with foodborne illnesses. Handling practices used during preparation of broiler chickens in 31 fast-food restaurants and 86 semisettled street stands (street vendors) were assessed by use of a standard checklist. These establishments used wood, plastic, or metal cutting surfaces during the preparation of broiler chickens. The survival of Campylobacter spp. on kitchen cutting surfaces was determined by inoculating approximately 10(6) CFU of Campylobacter jejuni onto sterile plastic, wooden, and metal cutting boards. The concentrations of the organisms were then assessed in triplicate on each type of cutting board over a 3-h period using standard microbiological methods for thermophilic Campylobacter spp. In 87% of food establishments, the same work area was used for preparation of raw and cooked chicken, and in 68% of these establishments the same cutting boards were used for raw and cooked chicken. None of the establishments applied disinfectants or sanitizers when washing contact surfaces. Campylobacter spp. survived on wooden and plastic but not on metal cutting boards after 3 h of exposure. The handling practices in food preparation areas, therefore, provide an opportunity for cross-contamination of Campylobacter spp. to ready-to-eat foods.     

PREVALENCE OF THERMOPHILIC CAMPYLOBACTER SPP. IN RETAIL CHICKEN MEAT IN ANKARA

From May to August 2004, 127 samples of chicken meat for sale on the retail market in Ankara were analyzed for the prevalence of thermophilic Campylobacter spp. Campylobacter spp. were isolated from 83.4% of the samples analyzed. Campylobacter jejuni was found in 74.8% of all samples. A total of 364 thermophilic Campylobacter strains were isolated and the species distribution among these strains was 70.1% C. jejuni, 21.1% Campylobacter coli and 8.6% Campylobacter lari. The results obtained from the study indicate that hygienic and technical compliance is needed in all stages of poultry processing to reduce contamination and to prevent public health hazard. An integrated HACCP plan must be applied from the farm to the table for the prevention of C. jejuni infections.     

Aeromonas spp. in foods: a significant cause of food poisoning?

From a total of 563 samples of various foodstuffs purchased from retail outlets in the Reading area 287 were found to contain mesophilic Aeromonas spp. The types of sample which were most frequently contaminated were poultry (79.3%) and offal (84.3%). Of three media compared for their efficiency in recovering Aeromonas spp. after enrichment in alkaline peptone water, Difco Aeromonas was the most efficient. Cytotoxin was produced by approximately 50% of the A. hydrophila and A. sobria strains but by none of A. caviae strains. It is concluded that both raw and cooked foods are potential sources for infecting human beings with Aeromonas spp.     

Evaluation of a commercial automated ELISA and PCR-method for rapid detection and identification of Campylobacter jejuni and C. coli in poultry products

Two rapid methods for detection and identification of Campylobacter jejuni andCampylobacter coli in naturally contaminated poultry products were evaluated for their applicability in routine studies. These methods were a commercial automated enzyme-linked immunosorbent assay (ELISA) for detection of thermophilic campylobacters in food, and C. jejuni -specific Polymerase chain reaction (PCR) analysis, which included a sample preparation method, based on Buoyant Density Centrifugation (BDC). Both methods were applied to the samples (97 samples) after a 48h enrichment procedure. The automated ELISA proved to be an easily repeatable and reliable method for detection of thermophilic campylobacters in food. The BDC-PCR method, which detected C. jejuni directly from the enrichment broth, was also reliable but not as simple as the ELISA. The analysis time for the ELISA-based method, starting from enrichment, took a minimum 2·5 working days, while the BDC-PCR method required 3 working days. Our study with a limited number of samples suggest that both detection methods are useful for monitoring C. jejuni on chicken meat.     

Investigation for possible source(s) of contamination of ready-to-eat meat products with Listeria spp. and other pathogens in a meat processing plant in Trinidad

In 2003, there was a recall of three processed (chicken franks, spice ham and turkey ham ready-to-eat (RTE) meat products by a large processing plant in Trinidad as a result of contamination by Listeria monocytogenes. The study was conducted to investigate the possible source(s) of Listeria contamination of recalled RTE meat products and to determine the prevalence of Listeria spp., Salmonella spp., Escherichia coli and Campylobacter spp. in the products and air within the plant. Raw and processed meat products, as well as food contact surfaces were also tested for Salmonella spp., Listeria spp. and Campylobacter spp. initially after thorough clean-up and close-down of the plant. Faecal and effluent samples from the piggery, in close proximity to the plant, were tested for the presence of Salmonella spp., Listeria spp. and Campylobacter spp. Air samples and food contact surfaces were negative for the tested organisms. Ten (58.8%) of the 17 effluent samples and 4 (11.8%) of the 34 faecal samples were positive for Campylobacter coli. Of the 11 raw meat products tested, 10 (90.9%) were positive for E. coli and Listeria spp. either singly or in combination. Of the 32 processed RTE products tested, 11 (34.4%) were positive for E. coli, Salmonella spp., Listeria spp. and Campylobacter spp. in combination or singly. Eleven (61.1%) of 18 processed products contained unacceptable levels of aerobic bacteria using international standards. Four months later, following the implementation of recommended cleaning, sanitizing and hygienic practices at the plant, pre- and post-processed products were sampled and Listeria spp. were identified in 4 (80.0%) of the 5 raw products and in 1 of the 5 (20.0%) finished products. Two (40.0%) of the finished products contained unacceptable microbial levels. It was concluded that the close proximity of the piggery to the processing plant was not the probable source of Listeria contamination of the recalled meat products. The data suggested that improved sanitary practices on food contact surfaces and during handling of products, reduced the risk of Listeria spp. and other pathogens studied. The problem at the plant can therefore, be inferred to be due to lapses in good sanitary practices, inadequate heat treatments or the presence of pathogens particularly Listeria in biofilms on different surfaces continuously or occasionally contaminating finished products.     

Diversity of Campylobacter isolates from retail poultry carcasses and from humans as demonstrated by pulsed-field gel electrophoresis

Campylobacter spp. are a major contaminant of poultry. Eating undercooked chicken and handling raw poultry have been identified as risk factors for campylobacteriosis in humans. Previous studies have found Campylobacter spp. on 90% of poultry carcasses. In the present study, pulsed-field gel electrophoresis (PFGE) was used to assess the genetic diversity of strains on retail poultry carcasses. PFGE patterns of isolates from campylobacteriosis cases were compared to those from the poultry isolates. Over a 1-year study period (March 2000 through February 2001), whole fresh young chickens (n = 72) were obtained from three retail outlets in an urban community in the south-central United States. Campylobacter spp. were isolated from 82% of these carcasses. Strains (n = 70) were defined on the basis of their PFGE pattern. Sixty-seven percent of the carcasses from which Campylobacter spp. were isolated were contaminated with more than one PFGE-distinguishable strain. During the 1-year study period, most of the PFGE patterns (59%) were limited to isolates obtained from a single carcass. Forty-one percent of the PFGE-distinguishable strains were recovered from more than one carcass. Ninety-seven percent of the carcasses contaminated with the same strain were purchased at the same time from the same store. To examine the degree of genetic stability, four strains were followed in vitro over an estimated 1,000 doublings. The PFGE pattern of one of these isolates underwent minor changes during in vitro growth. The data indicate extensive variability in the PFGE patterns of Campylobacter spp. isolated from humans and from poultry carcasses. In spite of difficulties caused by such diversity and the fact that some carcasses are contaminated with more than one strain, the pattern variation provides a useful method for linking a particular strain to its source.     

The influence of freezing and duration of storage on Campylobacter and indicator bacteria in broiler carcasses

In total, 215 commercially processed broiler carcasses were examined to determine optimum cultural enumeration, the effects of freezing, method of thawing, and duration of frozen storage on levels of Campylobacter spp. and fecal coliforms. Enumeration studies compared MPN procedures to direct plating onto selective mCCDA agar and indicated equivalency for quantitation of Campylobacter spp. Levels of Campylobacter and fecal coliforms were subsequently estimated by direct plating of carcass rinses. Freezing of naturally contaminated carcasses followed by storage at -20 degrees C for 31, 73, 122 and 220 days showed statistically significant (P< or =0.05) reductions in Campylobacter counts initially as compared with counts found on fresh product. Among 5 lots of broilers, levels of Campylobacter on carcasses were reduced by log mean values ranging from 0.65 to 2.87 after freezing and 31 days of storage. Similar reductions due to freezing were not observed for fecal coliforms counts. The level of Campylobacter was reduced by approximately one log immediately after freezing, and remained relatively constant during the 31-220 days of frozen storage. The levels were constant during 7 days of refrigerated storage. After 31 days of frozen storage there was a reduced rate in reduction of counts among broilers thawed at 7 degrees C as compared to thawing at 22 degrees C with either cultural method (MPN and mCCDA). These findings warrant consideration of the public health benefits related to freezing contaminated poultry prior to commercial distribution to reduce Campylobacter exposure levels associated with contaminated carcasses.     

Prevalence of Listeria monocytogenes and Salmonella in ready-to-eat food in Catalonia, Spain

Listeria monocytogenes and Salmonella are pathogenic bacteria that can contaminate food products during or after processing. Ready-to-eat (RTE) food does not undergo any treatment to ensure its safety before consumption, and therefore risk of foodborne disease must be considered if these pathogens are present in the food. To evaluate the prevalence of these pathogens in RTE food, 140 RTE fish product samples, 501 RTE meat product samples, 462 RTE dairy samples, and 123 RTE dishes and desserts, providing a total of 1,226 samples, were collected from retail stores and food industry and analyzed for the presence of L. monocytogenes. A total of 1,379 samples consisting of 187 RTE fish products and 569 RTE meat products, 484 RTE dairy products, and 139 RTE dishes and desserts were collected and analyzed for the presence of Salmonella. L. monocytogenes was isolated from 20% of frozen Atlantic bonito small pies, 7.9% of smoked salmon samples, 11.1% of the pork luncheon meat samples, 6.2% of frozen chicken croquettes, 16.9% of cured dried sausage samples, 12.5% of cooked ham samples, and 20% of cooked turkey breast samples. L. monocytogenes was also found to be present in 1.3% of fresh salty cheese samples and 15.1% of frozen cannelloni samples. Salmonella was isolated from 1.2% of smoked salmon samples, 1.5% of frozen chicken croquettes, 2% of cooked ham samples, and 11.1% of cured dried sausage samples. Overall, occurrence of these pathogens in RTE foods was similar to that previously reported in the literature.     

Development and application of oligonucleotide probes for in situ detection of thermotolerant Campylobacter in chicken faecal and liver samples

Based on Campylobacter 16S- and 23S-rRNA sequence data oligonucleotide probes specific for thermotolerant campylobacters and for members of the genus Campylobacter have been developed. The 16S-rRNA-targeted probe CAMP 653, recommended for a comprehensive detection of members of the genus Campylobacter, specifically detected all Campylobacter strains used in this study. Detection of thermotolerant species has been achieved by the 23S-rRNA-targeted probe CAJECO1427. Optimal hybridisation conditions have been derived for both probes from melting profiles of fluorescence-labelled probe-target hybrids recorded in fluorescence in situ hybridisation experiments (FISH). The FISH assay was evaluated both by spiking poultry faecal samples with Campylobacter jejuni and by detecting Campylobacter in naturally colonized chickens. C. jejuni was reliably detected at levels of 10(6) cfu/g faeces after a 3- h enrichment step in Blood Preston Selective broth. Low level contaminations (相似文献   

Quantitative risk assessment of Campylobacter spp. in poultry based meat preparations as one of the factors to support the development of risk-based microbiological criteria in Belgium

The objective of this study was to do an exercise in risk assessment on Campylobacter spp. for poultry based meat preparations in Belgium. This risk assessment was undertaken on the demand of the competent national authorities as one of the supportive factors to define risk-based microbiological criteria. The quantitative risk assessment model follows a retail to table approach and is divided in different modules. The contamination of raw chicken meat products (CMPs) was represented by a normal distribution of the natural logarithm of the concentration of Campylobacter spp. (ln[Camp]) in raw CMPs based on data from surveillance programs in Belgium. To analyse the relative impact of reducing the risk of campylobacteriosis associated with a decrease in the Campylobacter contamination level in these types of food products, the model was run for different means and standard deviations of the normal distribution of the ln[Camp] in raw CMPs. The limitation in data for the local situation in Belgium and on this particular product and more precisely the semi-quantitative nature of concentration of Campylobacter spp. due to presence/absence testing, was identified as an important information gap. Also the knowledge on the dose-response relationship of Campylobacter spp. was limited, and therefore three different approaches of dose-response modelling were compared. Two approaches (1 and 2), derived from the same study, showed that the reduction of the mean of the distribution representing the ln[Camp] in raw CMPs is the best approach to reduce the risk of Campylobacter spp. in CMPs. However, for the simulated exposure and approach 3 it was observed that the reduction of the standard deviation is the most appropriate technique to lower the risk of campylobacteriosis. Since the dose-response models used in approach 1 and 2 are based on limited data and the reduction of the mean corresponds with a complete shift of the contamination level of raw CMPs, demanding high efforts from the poultry industry, it is proposed to lower the standard deviation of the concentration of Campylobacter spp. in raw CMPs. This proposal corresponds with the elimination of the products that are highly contaminated. Simulation showed that eating raw chicken meat products can give rise to exposures that are 10(10) times higher than when the product is heated, indicating that campaigns are important to inform consumers about the necessity of an appropriate heat treatment of these type of food products.     

Campylobacter contamination during poultry slaughter in Belgium

The relation between internal carriage and surface contamination with thermophilic Campylobacter species in broilers was examined by molecular typing methods. Samples from 39 flocks were collected in three Belgian poultry slaughterhouses. From each flock, crop swabs before slaughter and intestines and neck skins during slaughter were collected. A total of 309 isolates were identified at species level and further characterized by flagellin gene A PCR/restriction fragment length polymorphism and pulsed-field gel electrophoresis. Isolates were identified as Campylobacter jejuni (90%), Campylobacter coli (8.7%), and Campylobacter lari (2.2%), and 27 genotypes could be distinguished by combining the two molecular methods. Seventy-two percent of the flocks arriving at the abattoir were colonized with campylobacters. After slaughter, 79% of the flocks had contaminated neck skins. In six flocks, genotypes isolated from the neck skins were also found in the alimentary tract from previously slaughtered flocks. Four of these flocks were initially free of Campylobacter. These four flocks might have had no contaminated carcasses after logistic slaughtering.     

2种检测方法对弯曲菌检出率的影响

目的比较国标法和双孔滤膜法在生禽肉中弯曲菌的检测率,探索生禽肉中弯曲菌分离鉴定的有效检测方法。方法随机采集冷冻和新鲜禽肉产品共102份,通过国标法和双孔滤膜法2种方法检测,观察2种方法从样品处理、增菌和分离培养过程中的不同,并比较2种方法检测弯曲菌检出率的差异。结果102份样品中国标法检出弯曲菌的检出率为28.43%,其中空肠弯曲菌和结肠弯曲菌的检出率分别为13.725%和14.705%;双孔滤膜法检出弯曲菌的检出率为53.92%,其中空肠弯曲菌和结肠弯曲菌的检出率分别为26.47%和27.45%;双孔滤膜法检出率显著高于国标法,差异有统计学意义(P<0.05)。结论应用双孔滤膜法能显著提高生禽肉食品中弯曲菌的检出率。     

2011—2016年广西壮族自治区市售肉及肉制品食源性致病菌污染状况分析

目的 了解广西壮族自治区市售肉及肉制品中食源性致病菌污染状况,为有效开展食源性疾病防控提供科学依据。方法 2011—2016年从广西壮族自治区14个市采集5类市售肉及肉制品共10 927份,按照国家标准方法进行9种食源性致病菌检验。结果 10 927份样品的食源性致病菌总检出率为5.0%(548/10 927),食源性致病菌检出率按样品种类依次为调理肉制品(33.3%,33/99)、生畜肉(24.5%,73/298)、生禽肉(24.2%,67/277)、冷冻肉糜制品(14.4%,14/97)、熟肉制品(3.6%,361/10 156)。检出的主要致病菌为沙门菌、金黄色葡萄球菌和单核细胞增生李斯特菌。生禽肉中未检出弯曲菌和致泻大肠埃希菌,调理肉制品中未检出致泻大肠埃希菌,熟肉制品中未检出志贺菌。熟肉制品各年度检出率范围为0.9%～4.9%。结论 广西壮族自治区市售肉及肉制品受到不同程度食源性致病菌污染,且污染持续多年存在。     

Occurrence of Campylobacter spp. in raw and ready-to-eat foods and in a Canadian food service operation

The occurrence of Campylobacter spp. in a variety of foods from Ottawa, Ontario, Canada, and raw milk samples from across Canada was determined over a 2-year period. The samples consisted of 55 raw foods (chicken, pork, and beef), 126 raw milk samples from raw milk cheese manufacturers, and 135 ready-to-eat foods (meat products, salads, and raw milk cheeses). Campylobacter jejuni was detected in 4 of the 316 samples analyzed: 1 raw beef liver sample and 3 raw chicken samples. An isolation rate of 9.7% was observed among the raw chicken samples tested. This study also investigated the role of cross-contamination in disseminating Campylobacter from raw poultry within a food service operation specializing in poultry dishes. Accordingly, kitchen surfaces within a restaurant in Ottawa, Ontario, were sampled between March and August 2001. Tests of the sampling method indicated that as few as 100 Campylobacter cells could be detected if sampling was done within 45 min of inoculation; however, Campylobacter spp. were not detected in 125 swabs of surfaces within the kitchens of this food service operation. Despite the reported high prevalence of Campylobacter spp. in raw poultry, this organism was not detected on surfaces within a kitchen of a restaurant specializing in poultry dishes.     

Contamination of Campylobacter spp. from foodstuff

Prevalence of Campylobacter spp. in foodstuff. All campylobacter spp. were identified as thermophilic campylobacter jejuni. campylobacter jejuni were most commonly isolated from crude birds samples (37%), both cooled and frozen. Campylobacter jejuni were recovered in 57% meat and semifinished items samples with contamination levels from the cooled samples of 270+/-470 colony forming units (cfu) per 100 g, frozen samples of 238+/-346 per 100 g and in 351% offal samples, with a contamination level from the cooled samples 100+/-360 per 100 g.     

Contamination of chicken carcasses in Gauteng, South Africa, by Salmonella, Listeria monocytogenes and Campylobacter

The presence of the foodborne pathogens, Salmonella spp., Listeria monocytogenes and Campylobacter spp., on 99 fresh and frozen chicken carcasses sourced from various retailers in Gauteng, South Africa, was investigated. Using culture methods, 60.6% of the carcasses were found to be contaminated with one or more pathogens, with 19.2%, 19.2% and 32.3% of the carcasses being found to harbour Salmonella, L. monocytogenes and Campylobacter, respectively. The extent of contamination with one or more pathogens was not significantly different (p>0.1) between fresh or frozen samples or between samples from butcheries, supermarkets or street vendors. Significantly more (p<0.1) fresh carcasses from butcheries than from other outlets were contaminated with Salmonella, while more fresh carcasses from supermarkets were contaminated with Campylobacter. The proportion of carcasses with L. monocytogenes from all sources were similar. Polymerase chain reaction (PCR) results indicate an even higher extent of pathogen contamination, but the PCR techniques need to be further refined before they can be used routinely.