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1.
1. The occurrence of Arcobacter spp. and three pathogenic species of Arcobacter from Iranian poultry carcasses was investigated at different steps of broiler processing to determine critical control points for reducing carcass contamination.

2. Samples were collected from (a) cloaca immediately before processing, (b) different points during processing and (c) at different stations in a processing plant of a slaughterhouse in southern Iran.

3. After enrichment steps in Arcobacter selective broth, DNA of the samples was extracted and three significant pathogen species of Arcobacter were identified based on polymerase chain reaction (PCR) detection of 16S rRNA and specific species PCR.

4. Out of a total of 540 samples, 244 (45%) were positive for Arcobacter spp. Arcobacter butzleri was more frequently detected (73% ± 13.9%) than A. cryaeophilus (9% ± 13.9%) and A. skirrowii (4.1%). In addition, co-colonisation (A. butzleri and A. cryaerophilus) occurred in 13.9% of the positive samples.

5. The results indicate a high prevalence of Arcobacter in the investigated slaughterhouse and broiler carcasses and that Arcobacter is not a normal flora of the broilers. Evidence for the presence of Arcobacter in the environment and water of processing plants suggests that these are sources of contamination of poultry carcasses. In addition, contamination of the poultry carcasses can spread between poultry meats in different parts and processes of the slaughterhouse (pre-scalding to after evisceration).  相似文献   


2.
《Veterinary microbiology》1997,57(4):325-336
Swine stomachs were surveyed for evidence of Arcobacter spp. and Helicobacter spp. infections associated with gastric ulceration. A nested PCR test targeted to the 16S rRNA was developed to detect many Arcobacter spp. and Helicobacter spp. An internal oligonucleotide probe was used for differentiation and confirmation of the PCR product. Tissue samples were obtained from the nonglandular and glandular regions of 86 swine stomachs. Evidence of infection with these microbes was detected in 51%, with 77% of the positive samples being identified as A. butzleri using a highly specific probe. Nonglandular stomach samples (44%) were more likely to be positive by PCR than samples from the glandular (23%) region. Gross lesions of any stage of gastric ulceration, ranging from parakeratosis, erosions and ulceration, were observed in 24% of stomachs examined. Of 21 samples with lesions, 52% were positive by the broadly reactive PCR assay for Arcobacter spp. and Helicobacter spp. The majority of PCR-positive samples (75%) had no gross lesions. When a single step PCR assay that was more specific for Arcobacter spp. was used on the nonglandular stomach samples, 10.4% of the 86 samples were positive. Arcobacter spp. were culture from four of the sample stomachs. Partial sequencing of the 16S rRNA gene identified the isolates as A. butzleri (n = 2), A. cryaerophilus, (n = 1), and a mixed culture of A. butzleri and another Arcobacter spp. (n = 1). A single step PCR assay targeted to the urease gene and culturing methods were used to screen for H. pylori or other closely related urease positive bacteria, but none were found.  相似文献   

3.
1. The objective of this study was to determine the prevalence and antimicrobial resistance of Arcobacter spp. isolated from different species of retail poultry meat in Iran.

2. From August 2012 to April 2013, a total of 540 raw poultry meat samples from chicken (n = 100), turkey (n = 100), quail (n = 100), partridge (n = 80), duck (n = 50), ostrich (n = 60) and geese (n = 50) were purchased from randomly selected retail outlets in Shahrekord, Isfahan, Sari and Rasht, Iran.

3. Using culture techniques, 71 of 540 poultry meat samples (13.1%) were positive for Arcobacter spp. The highest prevalence of Arcobacter spp. was found in chicken meat (28.0%), followed by quail (12.0%), duck (11.4%), turkey (11.0%), geese (8.0%), partridge (7.5%) and ostrich (3.3%) meat. The number of A. butzleri isolated from poultry meat samples (90.1%) was significantly higher than A. cryaerophilus (7.1%) and A. skirrowii (2.8%). Significantly more poultry meat samples were found to contain Arcobacter spp. by the PCR assay than by the culture method.

4. Susceptibilities of Arcobacter isolates were determined for 14 antimicrobial drugs using the disk diffusion method. All of the 71 Arcobacter isolates tested were resistant to one or more antimicrobial agents. Resistance to cephalothin and vancomycin (95.8%) was the most common finding, followed by resistance to methicillin, azithromycin and ampicillin. All Arcobacter isolates were susceptible to gentamicin, streptomycin, tetracyclin and kanamycin.

5. The results of this study indicated the importance of poultry meat, especially chicken meat, as potential sources of Arcobacter spp. infection in people. Furthermore, the strains indicated resistance to a broad spectrum of antibiotics.  相似文献   


4.
This study is conducted to determine the occurrence and antimicrobial resistance of Arcobacter spp. isolated from clinically healthy food animals. A total of 308 samples from cattle (200) and sheep (108) were collected from Shiraz slaughterhouse, southern Iran to investigate the presence of the important Arcobacter spp. using cultivation and Polymerase Chain Reaction (PCR) methods. Antimicrobial susceptibility of Arcobacter isolates was determined for 18 antibiotics using disk diffusion method. Among 308 samples, 27 (8.7%) and 44 (14.28%) were positive for the presence of Arcobacter species with cultivation and PCR procedures, respectively. The predominant species was A. butzleri in both cattle (58.33%) and sheep (55%). In addition, concurrent incidence of the species was observed in 25% of the positive samples. All Arcobacter isolates were resistant to rifampicin, vancomycin, ceftriaxone, trimethoprim and cephalothin. The isolates showed high susceptibility to tetracycline, oxytetracycline, erythromycin, ciprofloxacin, kanamycin, amikacin, gentamicin and enrofloxacin. No significant difference among cattle and sheep isolates in resistance pattern was observed. The results indicate that cattle and sheep are significant intestinal carriers for Arcobacter spp. Moreover, tetracycline and aminoglycosides showed great effects on Arcobacter species in antibiogram test and can be used for treatment of human Arcobacter infections.  相似文献   

5.
《Veterinary microbiology》1997,57(4):347-354
Seventeen field isolates of Arcobacter species were recovered in Brazil from aborted porcine fetal livers (n = 3), kidneys (n = 2), and thoracic fluid (n = 1). Arcobacter species were also recovered from uterine and oviductal tissues (n = 5) and a placenta from sows with reproductive problems. These isolates were initially presumed to be Arcobacter cryaerophilus on the basis of aerobic growth at 30°C, indoxyl acetate hydrolysis, catalase and oxidase reactions, growth on MacConkey agar, sensitivity to 3.5% sodium chloride, and susceptibility to nalidixic acid (40 mg/ml). The isolates were confirmed as Arcobacter using polymerase chain reaction, and were classified as A. cryaerophilus 1A (24%), A. cryaerophilus 1B (71%), and A. butzleri (6%) using restriction fragment length polymorphism.  相似文献   

6.
1. The frequency of thermophilic Campylobacter spp. on broiler carcases was determined during processing in a Southern Brazil slaughterhouse. Samples were collected after defeathering, evisceration, water chilling and freezing. In addition, samples were obtained from the water of the chiller tank and from the surface of equipment in direct contact with the chicken. 2. Samples (335) were analysed and 71.3% were positive for Campylobacter. The frequency of Campylobacter spp. on carcases rinsed in BPW and skin samples from carcases was 49 of 72 (68.0%) after defeathering, 50 of 72 (69.4%) after evisceration, 61 of 72 (84.7%) after chilling, and 46 of 72 (63.9%) after freezing. Campylobacter was positive for 21 of 23 (91.3%) samples in the chilling water and for 12 of 24 (50.0%) samples on the table surface. 3. The frequency of qualitative analysis for Campylobacter spp. was reduced in frozen chickens, but not during the slaughtering process. The use of drinking water alone as a decontaminant to reduce the incidence of Campylobacter spp. during slaughter is therefore not sufficient. Furthermore, to ensure food safety, chickens must be cooked properly before consuming.  相似文献   

7.
Campylobacter numbers on broiler carcasses can increase dramatically during defeathering because of leakage of contaminated gut contents in the feather-picking machine. Food-grade organic acids have been shown to be effective in killing bacteria. Placement of organic acids into the cloaca prior to defeathering was tested to determine if such a treatment could lower the number of Campylobacter that escape and contaminate broiler breast skin during automated feather removal. Campylobacter numbers on the breast skin of treated carcasses increased during defeathering but resulted in numbers that were only about 2% of those observed on control carcasses. Placement of food-grade organic acids in the cloaca of broiler carcasses may be useful as a means to lessen the impact of automated defeathering on the microbiological quality of carcasses during processing.  相似文献   

8.
The objective of this study was to develop a multiplex polymerase chain reaction (PCR) to detect and differentiate food‐borne pathogens of the three genera Campylobacter, Arcobacter and Helicobacter in a single step procedure. One common reverse primer and three genus‐specific forward primers were designed by hybridizing to the 16S rRNA of selected reference strains. Besides the species with significance as food‐borne pathogens isolated from poultry meat –Campylobacter jejuni, Campylobacter coli, Arcobacter butzleri and Helicobacter pullorum– several other members of these genera were tested to determine the specificity of the designed multiplex PCR. In total, 20 ATCC and NCTC reference strains of Campyobacter, Arcobacter and Helicobacter were used to evaluate the PCR. Specific amplificates were obtained from all thermophilic species of Campylobacter as well as from species of Arcobacter and Helicobacter. No amplification product was obtained from the non‐thermophilic Campylobacter, C. hyointestinalis and C. fetus. Furthermore, a total of 43 field strains of the three genera isolated from poultry, pigs, cattle and humans were investigated using this PCR. To confirm the classification of 10 H. pullorum strains the 16S rRNAs were sequenced. The developed PCR is a helpful diagnostic tool to detect and differentiate Campylobacter, Arcobacter and Helicobacter isolated from poultry and poultry products.  相似文献   

9.
In June and September 1988, the USDA Food Safety and Inspection Service sampled raw chicken carcasses at a federally inspected slaughter establishment in Puerto Rico to determine the effects of changing the scalding equipment on bacterial contents of raw poultry products. The scalding equipment was changed to a countercurrent configuration, with a postscald hot-water rinse cabinet that sprayed carcasses as they exited the scalder. Analysis of 250 carcass-rinse samples collected at preevisceration, prechill, and postchill sites over 7 days indicated that carcasses had mean aerobe plate counts of log(10)3.73 before evisceration, 3.18 before chilling, and 2.87 after chilling; Enterobacteriaceae counts of log(10)2.70 before evisceration, 2.25 before chilling, and 1.56 after chilling; and Escherichia coli counts of log(10)2.09 before evisceration, 1.61 before chilling, and 0.89 after chilling. Salmonellae were found on 24% of the carcasses before evisceration, on 28% before chilling, and on 49% after chilling. Although bacterial count reductions were significant at all 3 sites, the proportion of carcasses contaminated with salmonellae in this study was higher at the postchill than prechill site (49 vs 28%). This no doubt was caused by cross-contamination in the chiller. These percentages indicated that although simple scalder changes contributed substantially to the improvement of the bacterial quality of chicken carcasses, additional interventions in the chilling process (such as chlorination of chill water) are important to control cross-contamination and to preserve the positive effects obtained by the scalder changes.  相似文献   

10.
To assess post-evisceration contamination of broiler carcasses, 300 samples were randomly selected during routine slaughter in the winter of 2004. The samples originated from 50 chicken carcasses, taken directly after evisceration, as well as 25 samples from ready-to-sell packages of fresh intestines (mala) and livers. The samples were taken in batches over a period of 4 weeks to allow randomised sampling from different farms of origin. Conventional culture-based detection methods of Campylobacter spp. usually need 4-6 days to produce a result. The polymerase chain reaction (PCR) used for this study took less than 32 hours. The average contamination rates with Campylobacter in both the skin and liver samples were 24%, and 28% for intestines. Chicken and chicken products, especially livers and intestines, form an integral part of the traditional diet of many Black South Africans, as they are cheap and readily available in bulk and un-chilled for direct distribution, mainly through street vending and other informal retail outlets. This sudy showed that Campylobacter spp. are prevalent in poultry in South Africa. The handling of poultry meat and products contaminated with this organism in households and the potential for cross-contamination of other foods presents a high risk of infection to consumers in South Africa. The study also emphasised the need for further research in this field.  相似文献   

11.
The aim of this study was to perform the identification and molecular characterization of Arcobacter cryaerophilus and Arcobacter butzleri isolated from caiman (Caiman yacare), kept at a production farm, in Brazil. Forty fecal samples were analyzed. After isolation and identification, 21/40 strains of A. butzleri and 19/40 strains of A. cryaerophilus were subjected to PCR for potential virulence gene detection. The results of the PCR showed 38/40 strains positive for the cadF, cj1349, ciaB, and tlyA genes, 39/40 strains positive for the pldA gene, and 40/40 strains positive for the mviN gene. None of the strains presented the irgA gene. Hemagglutinin (hecA gene) and hemolysin (hecB) genes were detected in 21/40 and 16/40 strains, respectively. The SE-AFLP showed a great genetic diversity, but some clonally groups were disseminated in various tanks. These data reveal that the strains presented the same virulence traits described from Arcobacter isolated from food-borne disease in humans.  相似文献   

12.
Salmonella contamination of 71 chicken broiler flocks was investigated at the slaughterhouse in Reunion Island between October 2007 and January 2009. Samples were collected from live broiler chickens and chicken carcasses as well as the slaughterhouse environment. Salmonella spp. was isolated from 40 of 71 (56 % with a confidence interval 5 % [45–67]) broiler chicken flocks at slaughter. The most prominent serovars were Blockley (31 %), Typhimurium and Brancaster (14 %), Hadar (10 %), Salmonella multidrug resistant clinical organisms serotypes 1,4,[5],12:i:-, and Virchow (8 %) and Livingstone, St. Paul, Seftenberg, Llandoff, Infantis and Indiana. At the farm, 27 % of the broiler chicken flocks tested positive for Salmonella spp. Salmonella spp. was isolated from 124 of 497 environmental samples (25 %). In most cases, there was no relationship between pulsed field gel electrophoresis (PFGE) pattern and antibiotic resistance pattern. The predominant Salmonella serovars were susceptible to most of the tested antibiotic drugs, but S. Hadar exhibited multidrug resistance. This study highlighted the primary source of Salmonella was the farm of origin and downstream stages in processing could not remedy to but amplify this Salmonella contamination.  相似文献   

13.
The emerging foodborne and waterborne pathogen, Arcobacter, has been linked to various gastrointestinal diseases. Currently, 19 species are established or proposed; consequently, there has been an increase in the number of publications regarding Arcobacter since it was first introduced in 1991. To better understand the potential public health risks posed by Arcobacter, this review summarizes the current knowledge concerning the global distribution and the prevalence of Arcobacter in food and water. Arcobacter spp. were identified in food animals, food‐processing environments and a variety of foods, including vegetables, poultry, beef, dairy products, seafood, pork, lamb and rabbit. A wide range of waterbodies has been reported to be contaminated with Arcobacter spp., such as wastewater, seawater, lake and river water, drinking water, groundwater and recreational water. In addition, Arcobacter has also been isolated from pets, domestic birds, wildlife, zoo and farm animals. It is expected that advancements in molecular techniques will facilitate better detection worldwide and aid in understanding the pathogenicity of Arcobacter. However, more extensive and rigorous surveillance systems are needed to better understand the occurrence of Arcobacter in food and water in various regions of the world, as well as uncover other potential public health risks, that is antibiotic resistance and disinfection efficiency, to reduce the possibility of foodborne and waterborne infections.  相似文献   

14.
Abstract

AIM: To provide baseline data on the levels and patterns of antibacterial drug resistance expressed by Gram-negative bacteria isolated from poultry carcasses in New Zealand.

METHODS: Between July and December 2006, isolates of Escherichia coli (n=407) and Salmonella spp. (n=3) originating from carcass-rinse samples were submitted by testing laboratories affiliated to five major poultry processing plants. Isolates of Campylobacter jejuni (n=193) originating from retail poultry carcasses in 2005–2006 were retrieved from the Massey University archives. All isolates underwent disc diffusion susceptibility testing against panels of 12 (Enterobacteriaceae) and six (Campylobacter spp.) antibacterial drugs. Cephalothin-resistance in isolates of E. coli was confirmed using ETest strips, and confirmation of the resistance phenotypes for a subset of C. jejuni isolates used microbroth dilution assays. Patterns within the resistance phenotypes of the isolates were investigated using hierarchical clustering, and logistic regression modelling.

RESULTS: The majority of isolates (71.5% E. coli, 99% C. jejuni, and all three Salmonella spp. isolates) were fully susceptible to the drugs that were tested. Four (1%) E. coli isolates showed resistance to three or more drugs. The proportions of susceptible E. coli differed between the five processing plants. Resistances were detected in E. coli isolates, using disc diffusion to cephalothin (18.2%), ampicillin (4.4%), tetracycline (4.4%) and gentamicin (1.5%). There was an association between cephalothin-resistant isolates of E. coli and decreased susceptibility to gentamicin. Using ETests to ascertain the minimum inhibitory concentrations (MIC) of E. coli for cephalothin gave inconsistent results. One of 193 C. jejuni isolates was resistant to erythromycin, and microbroth dilution assays confirmed that this panel of C. jejuni was generally susceptible to antibacterial drugs.

CONCLUSIONS: The levels of resistance shown by Gram-negative bacteria isolated from chicken carcasses in New Zealand are among the lowest reported around the world. No resistance to extended-spectrum cephalosporin drugs was detected in E. coli, suggesting that CTX-M and AmpC beta-lactamases are rare or absent. Salmonella spp. are rarely isolated from poultry carcasses during routine testing in New Zealand, and the isolates identified during this study were fully susceptible to the drugs tested. A panel of C. jejuni isolates originating from retail poultry carcasses were susceptible to first-line and second-line antibacterial drugs. The use of cephalothin as a marker of resistance to first-generation cephalosporins may not be appropriate for non-type-specific E. coli of animal origin.  相似文献   

15.
Broiler meat is regarded as the most common source of Campylobacter infection and risk management measures are required to reduce broiler meat contamination. Among the quantitative risk assessments for Campylobacter in broiler meat, evaluation of the poultry processing stage is particularly important for predicting the contamination level of broiler meat and the effects of control measures. In this study, we built a simulation model for cross‐contamination during poultry processing focusing on Campylobacter contamination at the individual carcass level. Using this model, we examined changes in the prevalence of contaminated carcasses and the number of Campylobacter per carcass after processing. As a result, it was found that the prevalence and number of Campylobacter after processing were largely influenced by the number of Campylobacter on the contaminated carcasses before processing. In the baseline model, where it was assumed that the mean number of Campylobacter on contaminated carcasses before processing was 4.7 log10 cfu per carcass, the prevalence after processing was less than that before processing. Although the median value of Campylobacter on contaminated carcasses was reduced after processing, the distributions after processing became wider and the upper limit of the 95% credible interval of Campylobacter on contaminated carcasses remained elevated. The individual‐based simulation model can trace individual level changes considering discrete interactions, while models tracing mean values cannot handle these interactions in detail. The individual‐based approach is considered useful for modelling cross‐contamination among individual carcasses during poultry processing.  相似文献   

16.
1. Sampling carcasses after plucking or after the post‐evisceration spray‐wash showed that 10 or 20 ppm available chlorine in the processing‐plant water supply caused little reduction in carcass contamination.

2. When 20 ppm chlorine was used in the water supply to parts of the processing‐plant other than the mechanical immersion chilling system, counts of faecal and spoilage bacteria from carcasses were reduced approximately 10‐fold after passage through the chilling system; bacterial numbers were correspondingly decreased in the chiller water due to a carry‐over of chlorine from the final spray‐washer.

3. Artificial contamination of carcasses with a readily identifiable strain of Escherichia coli confirmed the occurrence of cross‐contamination during plucking and evisceration; in‐plant chlorination reduced neither the proportion of carcasses contaminated nor the numbers of organisms transferred at these stages.

4. In most cases the chlorine‐resistance of poultry spoilage pseudo‐monads was greater than that of E. coli; hence in‐plant chlorination is to be recommended for processing‐plant water supplies which carry such spoilage organisms.  相似文献   


17.
An observational study was conducted of chicken and turkey flocks slaughtered at federal processing plants in the province of Quebec, Canada. The objectives were to estimate prevalence of drug use at hatchery and on farm and to identify antimicrobial resistance (AMR) in cecal Escherichia coli and Enterococcus spp. isolates and factors associated with AMR. Eighty-two chicken flocks and 59 turkey flocks were sampled. At the hatchery, the most used antimicrobial was ceftiofur in chickens (76% of flocks) and spectinomycin in turkeys (42% of flocks). Virginiamycin was the antimicrobial most frequently added to the feed in both chicken and turkey flocks. At least 1 E. coli isolate resistant to third-generation cephalosporins was present in all chicken flocks and in a third of turkey flocks. Resistance to tetracycline, streptomycin, and sulfisoxazole was detected in > 90% of flocks for E. coli isolates. Antimicrobial resistance (AMR) was observed to bacitracin, erythromycin, lincomycin, quinupristin-dalfopristin, and tetracycline in both chicken and turkey flocks for Enterococcus spp. isolates. No resistance to vancomycin was observed. The use of ceftiofur at hatchery was significantly associated with the proportion of ceftiofur-resistant E. coli isolates in chicken flocks. In turkey flocks, ceftiofur resistance was more frequent when turkeys were placed on litter previously used by chickens. Associations between drug use and resistance were observed with tetracycline (turkey) in E. coli isolates and with bacitracin (chicken and turkey), gentamicin (turkey), and tylosin (chicken) in Enterococcus spp. isolates. Further studies are needed to provide producers and veterinarians with alternative management practices and tools in order to reduce the use of antimicrobial feed additives in poultry.  相似文献   

18.
An investigation was made of the contamination of poultry carcasses with clostridia during processing. In both chickens and turkeys clostridia were present initially on the feet and breast feathers (geometric mean counts of 102‐103/g) and were frequently isolated from the vent area immediately after evisceration. Mean counts from the breast surface just before packaging were < I‐3/10 cm2 and there were 31–467/g in samples of neck skin at the same stage. Counts were progressively lower in water samples taken from scald‐tanks, wash‐tanks, spin‐chillers and chill‐tanks.

A large proportion of the isolates obtained from both chicken and turkey plants were haemolytic Clostridium welchii, but a significant number of strains from the turkey plants could not be identified with recognised species.  相似文献   


19.
Workers in poultry abattoirs may be frequently exposed to Campylobacter jejuni, which is a leading cause of bacterial food poisoning in Japan. The present study was conducted to measure the titers of IgG and IgA antibodies against C. jejuni among 104 female workers in a chicken processing plant in Miyazaki prefecture, Japan. Information regarding habitual ingestion of raw chicken meat and potential occupational risk factors was collected using a questionnaire. Acid extracts of four C. jejuni strains representing the genotypes most dominant in Miyazaki were used as antigens. The levels of both immunoglobulins measured by ELISA were not correlated with ingestion of edible raw chicken meat, the amount consumed in one sitting, or its frequency. Although age was correlated with antibody levels, the length of employment was not. Furthermore, the IgG and IgA levels in workers at the evisceration step were significantly higher than those at other locations in the plant. To identify the bacterial proteins recognized by the workers’ IgG and IgA antibodies, Western blotting followed by LC/MS was conducted. Flagellin was identified as the common protein recognized in the sera of workers for whom ELISA demonstrated both the highest and lowest antibody levels. We concluded that the titers of IgG and IgA against C. jejuni in workers at the processing plant had been increased by occupational exposure to Campylobacter, regardless of raw chicken meat ingestion.  相似文献   

20.

Background

Microbiological standards within pork slaughter processing plants in the European Union are currently governed by Commission Regulation (EC) 2073/2005, which describes detailed performance criteria at specific stages of the procedure (following carcass dressing and before chilling) for total viable counts (TVC), Enterobacteriaceae (EB) and Salmonella spp. In this study, 95 carcasses from an Irish pork slaughter plant were sampled by swabbing 100 cm2 of surface at three sites (belly, ham, jowl) to examine the effects of eight processing stages (stunning, bleeding, scalding, singeing, polishing, evisceration, final inspection and chilling) on contamination levels.

Results

TVC ranged from approximately 1.7–6.3 log cfu cm2 during sampling. There were significant reductions in TVC for all sites after scalding and singeing (p < 0.05), whilst there was a significant increase in counts after polishing and evisceration (p < 0.05) compared with preceding stages. EB counts indicated hygienic weak points in the examined slaughter plant leading to faecal (cross)-contamination, with elevated counts after stunning, bleeding and evisceration (p < 0.05), compared with final counts after chilling.

Conclusions

Although the bacterial numbers reported in this study may reflect specific plant practices and temporal influences, results show that contamination can be introduced at various steps in the process and highlight the importance of monitoring locations other than those required by legislation within the process. Monitoring can be used to establish baseline levels for high-risk stages specific to each plant and to assess the effectiveness of additional interventions.  相似文献   

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