Comparison of Fluorescent-Antibody Methods and Enrichment Serology for the Detection of Salmonella

Four rapid methods for detection of Salmonella, (i) the conventional fluorescent-antibody (FA) technique, (ii) a rapid direct FA technique, (iii) microcolony FA, and (iv) enrichment serology (ES), were compared with conventional cultural procedures. A total of 347 subsamples representing 16 different food prototypes, alleged to be naturally contaminated with Salmonella, were analyzed. From these samples, 52 were found to contain Salmonella by cultural methods. Conventional FA identified all 52 culturally positive samples, ES identified 51, microcolony FA identified 48, and the rapid FA method identified 34. The number of false-positive samples for each procedure was: ES-selenite, 7; tetrathionate, 8; rapid FA, 26; microcolony FA, 33; conventional FA-selenite, 27; tetrathionate, 26. Tetrathionate enrichment was found to be superior to selenite for Salmonella recovery from most foods, but the concurrent use of both media allowed maximum recovery.     

Accelerated Immunofluorescence Procedure for the Detection of Salmonella in Foods and Animal By-Products

An accelerated, direct immunofluorescent-antibody procedure was developed for the detection of Salmonella in food products. This method includes pre-enrichment and selective enrichment but eliminates many of the washing and smear treatments present in existing methods. Commercially available fluorescein-conjugated somatic antiserum was used in comparing this method with conventional culture, biochemical, and serological procedures. The 894 samples tested represented 39 different products. The fluorescent-antibody procedure detected Salmonella in 216 test samples as compared to 205 positives recovered by using the standard culture procedures. In no instance did the fluorescent-antibody procedure fail to detect a Salmonella positive which had been detected by the standard procedure. With a three-tube, most-probable-number procedure, the fluorescent-antibody method was able to detect Salmonella at a level of 0.036 organism per g. In addition to being a more rapid method for the detection of Salmonella, it has proven to be comparable to conventional culture procedures.     

Accelerated Procedure for Salmonella Detection in Dried Foods and Feeds Involving Only Broth Cultures and Serological Reactions

A procedure has been developed in which Salmonella can be detected in dried foods and feeds within 50 hr. This includes preenrichment (18 hr), selective enrichment (24 hr), elective enrichment (6 to 8 hr), and serological testing (2 hr). The procedure is as sensitive as the more time-consuming, traditional (plating, biochemical, and serological) procedure which may require at least 4 to 5 days. The new procedure is rapid and accurate in comparison to traditional procedures. Moreover, it is simple and inexpensive to perform. As described, the procedure does not necessitate the isolation of pure cultures, but this step is performed easily if desired.     

Timed-Release Capsule Method for the Detection of Salmonellae in Foods and Feeds

A new method was developed for the detection of injured and uninjured salmonellae in foods and feeds. The steps of pre-enrichment in a nonselective broth and selective enrichment in a selective medium were combined into a single procedure. This was achieved by the gradual release of selective agents from wax-coated gelatin capsules added at the time of inoculation of nonselective basal broths. Pre-enrichment in lactose broth was combined with selective enrichment in tetrathionate or selenite-cystine broth by using timed-release capsules containing iodine or selenite. Five different categories of foods and feeds, naturally contaminated with salmonellae, were examined to compare the efficiencies of the capsule methods with conventional procedures. Combination of the separate steps of pre-enrichment and selective enrichment into a single procedure was feasible and resulted in substantial savings of labor and materials.     

Evaluation of a Fluorescent Antibody-Enrichment Serology Combination Procedure for the Detection of Salmonellae in Condiments, Food Products, Food By-Products, and Animal Feeds

The reliability of the enrichment serology (ES), fluorescent antibody (FA), and a combination of the FA and ES procedures for the detection of salmonellae were compared to the Salmonella cultural procedure outlined in the U.S. Food and Drug Administration's Bacteriological Analytical Manual (BAM). A total of 126 subsamples from 22 different products were analyzed. By utilizing the BAM procedure as the reference standard, a total of 66 samples were positive for salmonellae. Within 44 h approximately 65% of the Salmonella-negative samples could be cleared by the FA test. At the end of 50 h 97% of the Salmonella-negative samples could be cleared by the combination FA-ES test. The FA procedure detected all 66 BAM positives but exhibited a high incidence of presumptive positives which were cultural negatives. The ES procedure detected 64 of the 66 BAM positives but exhibited a low incidence of presumptive positives which were cultural negatives. Incorporating positive FA and positive ES results in a combination FA-ES technique revealed that FA-ES positives were statistically equivalent to BAM positives.     

Rapid and Specific Detection of Salmonella spp. in Animal Feed Samples by PCR after Culture Enrichment

A PCR procedure has been developed for routine analysis of viable Salmonella spp. in feed samples. The objective was to develop a simple PCR-compatible enrichment procedure to enable DNA amplification without any sample pretreatment such as DNA extraction or cell lysis. PCR inhibition by 14 different feed samples and natural background flora was circumvented by the use of the DNA polymerase Tth. This DNA polymerase was found to exhibit a high level of resistance to PCR inhibitors present in these feed samples compared to DyNAzyme II, FastStart Taq, Platinum Taq, Pwo, rTth, Taq, and Tfl. The specificity of the Tth assay was confirmed by testing 101 Salmonella and 43 non-Salmonella strains isolated from feed and food samples. A sample preparation method based on culture enrichment in buffered peptone water and DNA amplification with Tth DNA polymerase was developed. The probability of detecting small numbers of salmonellae in feed, in the presence of natural background flora, was accurately determined and found to follow a logistic regression model. From this model, the probability of detecting 1 CFU per 25 g of feed in artificially contaminated soy samples was calculated and found to be 0.81. The PCR protocol was evaluated on 155 naturally contaminated feed samples and compared to an established culture-based method, NMKL-71. Eight percent of the samples were positive by PCR, compared with 3% with the conventional method. The reasons for the differences in sensitivity are discussed. Use of this method in the routine analysis of animal feed samples would improve safety in the food chain.     

丙型肝炎病毒准种血清学检测技术的建立

目的:建立一种以血清学为基础的丙型肝炎病毒(HCV)准种检测技术。方法:自20份HCV血清中各挑选30个克隆进行测序比较,分析HCV准种的复杂程度;以HCV准种代表性抗原组合制备免疫芯片,用血清学检测技术分析上述20份HCV血清中的准种变异程度;比较两种方法之间的检出灵敏度和相关性。结果:测序法检出灵敏度为70.0%,血清学检测法检出灵敏度为95.0%,后者显著高于前者(P0.05);两种方法检测结果的相关性为74.7%(P0.01)。结论:血清学检测技术操作简单,且能够反映丙型肝炎患者的HCV准种变异程度,适于临床推广。     

The Fluorescent Antibody Technique for the Detection of Salmonella in Routine Use

S ummary : The direct and indirect fluorescent antibody technique (FAT) were compared with cultural methods for detecting salmonellae in meat products, animal feedingstuffs, poultry carcase swabs, giblets and poultry plant and equipment swabs. Salmonellae were not isolated from meat products and fluorescent cells were not seen on slides prepared by either FAT. The indirect and direct FAT recorded 13% and 9% respectively, false positive results, with samples of animal feedingstuffs, but the direct FAT recorded a single false negative result. Salmonellae were not isolated from poultry carcase swabs but 3% and 4·5% respectively, of false positive results were obtained with the indirect and direct FAT. Salmonellae were isolated from both giblet samples and poultry plant swabs and both gave rise to false negative FAT results. Preliminary studies of the efficacy of the FAT for screening animal faecal material for salmonellae indicated that no single combination of enrichment broth and FAT gives unequivocal results, but the staining of smears from tetrathionate broth by either FAT gives rise to a high percentage of false negative results.     

A Real-Time PCR Assay for the Detection of Campylobacter jejuni in Foods after Enrichment Culture

A real-time PCR assay was developed for the quantitative detection of Campylobacter jejuni in foods after enrichment culture. The specificity of the assay for C. jejuni was demonstrated with a diverse range of Campylobacter species, related organisms, and unrelated genera. The assay had a linear range of quantification over six orders of magnitude, and the limit of detection was approximately 12 genome equivalents. The assay was used to detect C. jejuni in both naturally and artificially contaminated food samples. Ninety-seven foods, including raw poultry meat, offal, raw shellfish, and milk samples, were enriched in blood-free Campylobacter enrichment broth at 37°C for 24 h, followed by 42°C for 24 h. Enrichment cultures were subcultured to Campylobacter charcoal-cefoperazone-deoxycholate blood-free selective agar, and presumptive Campylobacter isolates were identified with phenotypic methods. DNA was extracted from enrichment cultures with a rapid lysis method and used as the template in the real-time PCR assay. A total of 66 samples were positive for C. jejuni by either method, with 57 samples positive for C. jejuni by subculture to selective agar medium and 63 samples positive in the real-time PCR assay. The results of both methods were concordant for 84 of the samples. The total time taken for detection from enrichment broth samples was approximately 3 h for the real-time PCR assay, with the results being available immediately at the end of PCR cycling, compared to 48 h for subculture to selective agar. This assay significantly reduces the total time taken for the detection of C. jejuni in foods and is an important model for other food-borne pathogens.     

Efficiency of Selenite Cystine and TT Enrichment Broths for the Detection of Salmonella

Comparisons of selenite cystine (SC) and TT enrichment broths for detecting salmonellas were made with pure culture suspensions, with samples of naturally or artificially contaminated foods and with poultry feed. Selenite cystine recovered higher numbers of salmonellas from pure cultures and ground beef, while TT broth recovered higher numbers from pork sausage and poultry feed. Differences in the recovery of salmonellas from other food products appeared to be insignificant. The use of both SC and TT is thus recommended for maximal recovery of these organisms.     

A Rapid, Presumptive Procedure for the Detection of Salmonella in Foods and Food Ingredients

A rapid detection procedure was developed in which a lysine-iron-cystine-neutral red (LICNR) broth medium, originally described by Hargrove et al. in 1971, was modified and used to detect the presence of viable Salmonella organisms in a variety of foods, food ingredients, and feed materials by using a two-step enrichment technique. Tetrathionate broth was used to enrich samples with incubation at 41 C for 20 hr, followed by transfer to LICNR broth and incubation at 37 C for 24 hr for further enrichment and for the detection of Salmonella organisms by color change. One hundred ten samples representing 18 different sample types were evaluated for the presence of viable Salmonella. Ninety-four percent of the samples found to be presumptive positive by this method were confirmed as positive by a culture method. Fluorescent-antibody results also compared closely. A second study was conducted under quality-control laboratory conditions by using procedures currently employed for Salmonella detection. One hundred forty-three samples representing 19 different sample types were evaluated for the presence of viable Salmonella. No false negatives were observed with the rapid-detection method. The usefulness of the LICNR broth procedure as a screening technique to eliminate negative samples rapidly and to identify presumptive positive samples for the presence of viable Salmonella organisms was established in this laboratory.     

应用不同方法检测食品中大肠菌群结果的比较

评价检测食品中大肠菌群不同方法。比较国家标准、行业标准和显色培养基检测方法检测大肠菌群结果的差别。国家标准和行业标准检测结果基本一致,但有差异,应用显色培养基检测大肠菌群优于目前使用的国家标准和出口食品检验行业标准方法。检测食品中大肠菌群,显色培养基检测方法快速、灵敏、特异。     

Human Salmonella Clinical Isolates Distinct from Those of Animal Origin

The global trend toward intensive livestock production has led to significant public health risks and industry-associated losses due to an increased incidence of disease and contamination of livestock-derived food products. A potential factor contributing to these health concerns is the prospect that selective pressure within a particular host may give rise to bacterial strain variants that exhibit enhanced fitness in the present host relative to that in the parental host from which the strain was derived. Here, we assessed 184 Salmonella enterica human and animal clinical isolates for their virulence capacities in mice and for the presence of the Salmonella virulence plasmid encoding the SpvB actin cytotoxin required for systemic survival and Pef fimbriae, implicated in adherence to the murine intestinal epithelium. All (21 of 21) serovar Typhimurium clinical isolates derived from animals were virulent in mice, whereas many (16 of 41) serovar Typhimurium isolates derived from human salmonellosis patients lacked this capacity. Additionally, many (10 of 29) serovar Typhimurium isolates derived from gastroenteritis patients did not possess the Salmonella virulence plasmid, in contrast to all animal and human bacteremia isolates tested. Lastly, among serovar Typhimurium isolates that harbored the Salmonella virulence plasmid, 6 of 31 derived from human salmonellosis patients were avirulent in mice, which is in contrast to the virulent phenotype exhibited by all the animal isolates examined. These studies suggest that Salmonella isolates derived from human salmonellosis patients are distinct from those of animal origin. The characterization of these bacterial strain variants may provide insight into their relative pathogenicities as well as into the development of treatment and prophylactic strategies for salmonellosis.     

Deprivation and Enrichment in Laboratory Animal Environments

Resistance in the horse to trailer loading is a common source of stress and injury to horses and their handlers. The objective of this study was to determine whether nonaversive training based on the Tellington-Touch Equine Awareness Method (TTEAM; Tellington-Jones &; Bruns, 1988) would decrease loading time and reduce stress during loading for horses with a history of reluctance to load. Ten horses described by their owners as "problem loaders" were subjected to pretraining and posttraining assessments of loading. Each assessment involved two 7-min loading attempts during which heart rate and saliva cortisol were measured. The training consisted of six 30-min sessions over a 2-week period during which the horse and owner participated in basic leading exercises with obstacles simulating aspects of trailering. Assessment showed heart rate and saliva cortisol increased significantly during loading as compared to baseline (p < .001 and p < .05, respectively). Reassessment after training showed a decrease in loading time (p < .02), reduced heart rate during loading (p < .002), and reduced saliva cortisol as compared to pretraining assessments. Seven "good loaders" also were subject to loading assessment for physiological comparison. Increases in heart rate during loading were significantly higher in the good loaders (p < .001). Nonaversive training simulating aspects of loading may effectively reduce loading time and stress during loading for horses with a history of resistance to trailer loading.     

生物技术在食品检测方面的应用

综述了DNA探针、PCR、生物芯片、胶体金免疫层析及ELLSA等生物技术的基本原理及其在食品检测方面的应用。     

Enrichment Medium for Selection of Salmonella from Fish Homogenate

A new liquid medium, called “dulcitol selenite enrichment,” has been developed for the detection and enumeration of Salmonella in foods. The medium is not only highly selective in enriching Salmonella and inhibiting completely or appreciably other extraneous organisms commonly found in seafoods, but is also highly sensitive in recovering as low as 2 to 7 cells of Salmonella, even in the presence of large numbers (10⁴ to 10⁶ cells) of mixed flora common to these foods. The addition of seafood material does not seem to interfere with the sensitivity, selectivity, or productivity of the medium. Even physiologically debilitated cells of Salmonella were enriched well enough in this medium to be detected easily.     

Application of Rapid Dot Blot Immunoassay for Detection of Salmonella enterica Serovar Enteritidis in Eggs, Poultry, and Other Foods

Salmonella enterica serovar Enteritidis was detected in artificially inoculated eggs within 24 h through a rapid monoclonal antibody-based dot blot immunoassay. Detection in poultry and other products required 28 h. Samples were directly enriched in homogenized egg without the need for pre- or postenrichment steps. Serovar Enteritidis was detected in the presence of other bacteria when outcompeted 1:400.     

Nalidixic Acid for Enrichment of Auxotrophs in Cultures of Salmonella typhimurium

Nalidixic acid kills only growing cells of Salmonella typhimurium and can be used to enrich for auxotrophs in populations where prototrophs predominate.