Detection of Salmonella Strains and Escherichia coli O157:H7 in Feces of Small Ruminants and Their Isolation with Various Media

Salmonella strains and Escherichia coli O157:H7 were detected in 17 and 5 small ruminants in Virginia, respectively, of 287 tested. Background microflora interfered with the fecal analysis. The combination of Salmonella enzyme immunoassay (EIA) detection and xylose-lysine-deoxycholate agar isolation was satisfactory. Modifying enrichment to a 1:100 dilution enabled effective E. coli O157:H7 detection by EIA and isolation by sorbitol-MacConkey agar with cefixime-tellurite.     

Reproducibility of Positive Results for the Detection of Serum Galactomannan by Platelia™ Aspergillus EIA

Galactomannan (GM) was recently included in consensus guidelines as an indirect mycological criterion for the diagnosis of invasive aspergillosis. Currently, there is an enzyme immunoassay available to detect GM in biological samples, the Platelia? Aspergillus EIA. In this study, the reproducibility of positive results obtained using this assay was evaluated using serum samples from neutropenic patients. A trend toward lower values was observed, and 55 %(27/49) of positive results were negative after retesting. A low reproducibility of positive results for the detection of GM in serum was observed.     

Phagomagnetic immunoassay for the rapid detection of Salmonella

This work explores the use of the phage P22 in a phagomagnetic immunoassay for the rapid detection of Salmonella. The covalent attachment of wild-type phages was performed on two different magnetic carriers: carboxyl-activated magnetic nanoparticles (300 nm) and tosyl-activated magnetic microparticles (2.8 μm). The bacteria were captured and preconcentrated by the phage-modified magnetic particles, followed by the detection using specific anti-Salmonella antibodies conjugated to horseradish peroxidase as an optical reporter. Outstanding selectivity and sensitivity was obtained with this approach, achieving detection limits of 19 CFU mL^?1 in 2.5 h without any pre-enrichment, in milk samples. Moreover, if the samples were pre-enriched for 6 h, the method was able to detect as low as 1.4 CFU in 25 mL of milk. Therefore, the proposed strategy based on the combined use of phagomagnetic separation with immunological labeling is promising as a rapid and simple method for food safety.     

Immunofluorescence technique for the detection of salmonellae in various foods

Salmonella species have been detected in nine food varieties by use of fluorescent antibodies without false-positive or false-negative results. Test antisera were specially prepared, commercially available, conjugated polyvalent O globulin absorbed with cultures of Escherichia coli and Citrobacter freundii, and polyvalent phase II H globulin antibodies. Use of this technique permits a decrease of 24 hr in time normally required for Salmonella detection when compared with cultural Salmonella recovery methods.     

Sensitive enzyme immunoassay for the rapid diagnosis of influenza a virus infections in clinical specimens

Samples of nasopharyngeal secretion (NPS) from 100 infants and small children admitted for acute respiratory disease during the period from January to March 1989 were examined for the presence of influenza A virus. All samples were tested by enzyme immunoassay (EIA), fluorescent antibody (FA) technique and by isolation in cell culture 3–6 h after they were obtained from the patients. Of 24 influenza strains found by isolation, 21 were detected by EIA and 19 were FA⁺. In comparison with virus isolation, EIA gave the following values: sensitivity 88 %, specificity 100 %, positive prognostic value (PPV) 100 %, and negative prognostic value (NPV) 96 %. A rabbit anti-influenza-A serum (A-13) was used as catching antibody and a monoclonal anti-influenza-A pool against NP protein was used as detector antibody in EIA. A-13 gave bands corresponding to influenza A core proteins (NP and M1) in Western blot (WB) studies when different H3N2 strains were employed as antigens. A-13 gave only a band corresponding to the NP protein when H1N1 strains were examined by WB. The detection level by EIA for both H3N2 and H1N1 strains precipitated by polyethylene glycol from tissue culture maintenance medium was 1–2 ng.     

Domestic Mite Antigens in Floor and Airborne Dust at Workplaces in Comparison to Living Areas: A New Immunoassay to Assess Personal Airborne Allergen Exposure

Objectives

Allergens produced by domestic mites (DM) are among the most common allergic sensitizers and risk factors for asthma. To compare exposure levels between workplaces and living areas a new assay able to measure airborne DM antigen concentrations was developed.

Methods

At workplaces and in living areas, 213 floor dust samples and 92 personal inhalable dust samples were collected. For sensitive quantification of DM antigens, a new enzyme immunoassay (EIA) based on polyclonal antibodies to Dermatophagoides farinae extract was developed. Reactivity of five house dust mite and four storage mite species was tested. All dust samples were tested with the new EIA and with the Der f 1 and Der p 1-EIAs (Indoor Biotechnologies, UK) which detect major allergens from D. farinae and D. pteronyssinus by monoclonal antibodies. Samples below the detection limit in the DM-EIA were retested in an assay variant with a fluorogenic substrate (DM-FEIA).

Results

The newly developed DM-EIA detects antigens from all nine tested domestic mite species. It has a lower detection limit of 200 pg/ml of D.farinae protein, compared to 50 pg/ml for the DM-FEIA. DM antigens were detected by DM-EIA/FEIA in all floor dust and 80 (87%) of airborne samples. Der f 1 was found in 133 (62%) floor dust and in only 6 airborne samples, Der p 1 was found in 70 (33%) of floor samples and in one airborne sample. Der f 1 and DM concentrations were highly correlated. DM-antigens were significantly higher in inhalable airborne samples from textile recycling, bed feather filling, feed production, grain storage and cattle stables in comparison to living areas.

Conclusions

A new sensitive EIA directed at DM antigens was developed. DM antigen quantities were well correlated to Der f 1 values and were measurable in the majority (87%) of airborne dust samples. Some workplaces had significantly higher DM antigen concentrations than living areas.     

Development of a real-time multiplex PCR assay for the detection of multiple <Emphasis Type="Italic">Salmonella</Emphasis> serotypes in chicken samples

Background  

A real-time multiplex PCR assay was developed for the detection of multiple Salmonella serotypes in chicken samples. Poultry-associated serotypes detected in the assay include Enteritidis, Gallinarum, Typhimurium, Kentucky and Dublin. The traditional cultural method according to EN ISO 6579:2002 for the detection of Salmonella in food was performed in parallel. The real-time PCR based method comprised a pre-enrichment step in Buffered Peptone Water (BPW) overnight, followed by a shortened selective enrichment in Rappaport Vasilliadis Soya Broth (RVS) for 6 hours and subsequent DNA extraction.     

Molecular identification of Salmonella isolates from poultry and meat productsin Irbid City,Jordan

The sensitivity and accuracy of molecular diagnosis of Salmonella from meat and poultry products using polymerase chain reaction (PCR) was compared with conventional microbiological methods. A total of 212 samples representing the most frequently used fresh and frozen meat and poultry products (whole, cut, ground, and processed) were collected from different locations within the city of Irbid. DNA was extracted directly from each food sample and amplified using Salmonella-specific primers. Samples were also analysed using conventional microbiological methods for the presence of Salmonella spp. Results showed that Salmonella was detected in 185 samples out of 212 (87%) by PCR technique, while 172 (81%) samples were detected Salmonella positive by conventional microbiological methods. On the other hand, 27 (12.7%) samples were negative by PCR and 40 (18.8%) samples were negative by conventional microbiological methods. PCR assay proved to be an effective method for Salmonella detection in meat and poultry products with high specificity and sensitivity and more importantly a less time-consuming procedure. Using PCR, Salmonella spp. detection could be achieved within 24–36 h compared to 3–8 days for the conventional microbiological methods.     

Development and validation of an enzyme immunoassay for progesterone in bovine milk or blood plasma

We have developed and validated a sensitive, simple and direct (i.e. without extraction) enzyme immunoassay (EIA) system for the measurement of progesterone in bovine milk and blood plasma. Progesterone (P) has been analysed by a microtiterplate EIA, employing polyclonal antibodies against P-7α-carboxyethylthioether-BSA as the antigen. The enzyme used as a label is horseradish peroxidase (HRP) and the chromogen is tetramethylbenzidine (TMB). Sensitivity of the EIA has been greatly improved by introduction of a heterologous tracer, in which progesterone is coupled to HRP at the 6β position. Compared to the radioimmunoassay (RIA) in which the same antiserum has been used, the sensitivity is 20 times greater. The detection limit of the assay is 0.4 pg per well. The working range of the standard curve is 0–20 pg per well (i.e. 0–40 ng per ml), and 50% reduction of the initial binding is obtained with 2.5-5.0 pg. Results can be obtained either by spectrophotometric measurement at 450 nm, or by naked eye. Total time needed for the assay of 40 replicate samples is approximately 3 h. Comparison of the EIA system with a previously validated RIA system gave a regression line EIA = 0.85 RIA + 2.11 (r = 0.93, n = 400 milk samples). Application of the milk-progesterone EIA to pregnancy testing (n = 66) gave an accuracy of 79.6% for positive diagnoses and 100% for negative diagnoses.     

Diagnostic Real-Time PCR for Detection of Salmonella in Food

A robust 5′ nuclease (TaqMan) real-time PCR was developed and validated in-house for the specific detection of Salmonella in food. The assay used specifically designed primers and a probe target within the ttrRSBCA locus, which is located near the Salmonella pathogenicity island 2 at centisome 30.5. It is required for tetrathionate respiration in Salmonella. The assay correctly identified all 110 Salmonella strains and 87 non-Salmonella strains tested. An internal amplification control, which is coamplified with the same primers as the Salmonella DNA, was also included in the assay. The detection probabilities were 70% when a Salmonella cell suspension containing 10³ CFU/ml was used as a template in the PCR (5 CFU per reaction) and 100% when a suspension of 10⁴ CFU/ml was used. A pre-PCR sample preparation protocol including a preenrichment step in buffered peptone water followed by DNA extraction-purification was applied when 110 various food samples (chicken rinses, minced meat, fish, and raw milk) were investigated for Salmonella. The diagnostic accuracy was shown to be 100% compared to the traditional culture method. The overall analysis time of the PCR method was approximately 24 h, in contrast to 4 to 5 days of analysis time for the traditional culture method. This methodology can contribute to meeting the increasing demand of quality assurance laboratories for standard diagnostic methods. Studies are planned to assess the interlaboratory performance of this diagnostic PCR method.     

The Moroccan Food Snail, Helix aspersa, as a Source of Salmonella

A total of 270 samples, nine lots of 30 samples each, of imported Moroccan food snails was examined for the presence of Salmonella. Eighty-four samples (an overall incidence of 31.11%) and all nine lots contained Salmonella. No significant difference (P > 0.25) in the number of positive samples was observed by using either selenite cystine broth or tetrathionate broth when the samples had been pre-enriched in lactose broth. When used as direct selective enrichments with samples not pre-enriched in lactose broth, tetrathionate broth was significantly (P < 0.05) more productive than selenite cystine broth. The overall detection of Salmonella-positive samples by direct enrichment was significantly greater (P < 0.001) than by pre-enrichment. A variety of uncommon serotypes representative of several somatic groups was isolated. This study reports the occurrence and incidence, and the concomitant human health potential, of Salmonella in one species of live, imported food snails.     

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.     

Development and Evaluation of a Blood Culture PCR Assay for Rapid Detection of Salmonella Paratyphi A in Clinical Samples

Background

Enteric fever remains an important cause of morbidity in many low-income countries and Salmonella Paratyphi A has emerged as the aetiological agent in an increasing proportion of cases. Lack of adequate diagnostics hinders early diagnosis and prompt treatment of both typhoid and paratyphoid but development of assays to identify paratyphoid has been particularly neglected. Here we describe the development of a rapid and sensitive blood culture PCR method for detection of Salmonella Paratyphi A from blood, potentially allowing for appropriate diagnosis and antimicrobial treatment to be initiated on the same day.

Methods

Venous blood samples from volunteers experimentally challenged orally with Salmonella Paratyphi A, who subsequently developed paratyphoid, were taken on the day of diagnosis; 10 ml for quantitative blood culture and automated blood culture, and 5 ml for blood culture PCR. In the latter assay, bacteria were grown in tryptone soy broth containing 2.4% ox bile and micrococcal nuclease for 5 hours (37°C) before bacterial DNA was isolated for PCR detection targeting the fliC-a gene of Salmonella Paratyphi A.

Results

An optimized broth containing 2.4% ox bile and micrococcal nuclease, as well as a PCR test was developed for a blood culture PCR assay of Salmonella Paratyphi A. The volunteers diagnosed with paratyphoid had a median bacterial burden of 1 (range 0.1–6.9) CFU/ml blood. All the blood culture PCR positive cases where a positive bacterial growth was shown by quantitative blood culture had a bacterial burden of ≥ 0.3 CFU/ ml blood. The blood culture PCR assay identified an equal number of positive cases as automated blood culture at higher bacterial loads (≥0.3 CFU/ml blood), but utilized only half the volume of specimens.

Conclusions

The blood culture PCR method for detection of Salmonella Paratyphi A can be completed within 9 hours and offers the potential for same-day diagnosis of enteric fever. Using 5 ml blood, it exhibited a lower limit of detection equal to 0.3 CFU/ml blood, and it performed at least as well as automated blood culture at higher bacterial loads (≥0.3 CFU/ml blood) of clinical specimens despite using half the volume of blood. The findings warrant its further study in endemic populations with a potential use as a novel diagnostic which fills the present gap of paratyphoid diagnostics.     

Enumeration of Salmonella and Campylobacter spp. in Environmental Farm Samples and Processing Plant Carcass Rinses from Commercial Broiler Chicken Flocks

A prospective cohort study was performed to evaluate the prevalences and loads of Salmonella and Campylobacter spp. in farm and processing plant samples collected from 55 commercial broiler chicken flocks. Environmental samples were collected from broiler houses within 48 h before slaughter, and carcass rinses were performed on birds from the same flocks at 4 different stages of processing. Salmonella was detected in farm samples of 50 (90.9%) flocks and in processing samples of 52 (94.5%) flocks. Campylobacter was detected in farm samples of 35 (63.6%) flocks and in processing samples of 48 (87.3%) flocks. There was a significant positive relationship between environmental farm samples and processing plant carcass rinses with respect to both Salmonella and Campylobacter prevalences and loads. Campylobacter loads were significantly higher than Salmonella loads, and the correlations between samples collected from the same flocks were higher for Campylobacter than they were for Salmonella. Boot socks were the most sensitive sample type for detection of Salmonella on the farm, whereas litter samples had the strongest association with Salmonella loads in pre- and postchill carcass rinses. Boot socks, drag swabs, and fecal samples all had similar sensitivities for detecting Campylobacter on the farm, and all were more strongly associated with Campylobacter loads in carcass rinses than were litter samples. Farm samples explained a greater proportion of the variability in carcass rinse prevalences and loads for Campylobacter than they did for Salmonella. Salmonella and Campylobacter prevalences and loads both decreased significantly as birds progressed through the processing plant.     

Twelve-Hour PCR-Based Method for Detection of Salmonella spp. in Food

A PCR-based method for the detection of Salmonella spp. in food was developed. The method, set up on typical salami from the Italian region of Marche, is sensitive and specific and shows excellent correlation with the conventional method of reference when naturally contaminated foods are analyzed. Moreover, it can be easily performed within a maximum of 12 h from food sampling, thus allowing prompt detection of Salmonella spp. in the food stocks analyzed.     

Preparation and characterization of monoclonal antibodies to the cholera toxin

Monoclonal antibodies to cholera toxin were obtained. They do not cross-react with the termolabile toxin (LT) of Escherichia coli, ricin, diphtherial toxin, staphylococcus enterotoxins of SEA, SEB, SEI, SEG, or the lethal factor and protective antigen of the anthrax toxin. Pairs of antibodies for the quantitative measurement of the cholera toxin in sandwich enzyme immunoassay (EIA) were selected. The detection limit of the toxin is 0.2 ng/ml for plate EIA and 0.44 ng/ml for microchip EIA. The presence of milk, broth, or surface water in the toxin samples does not reduce the sensitivity of EIA.     

Detection of Salmonella enterica in Naturally Contaminated Liquid Eggs by Loop-Mediated Isothermal Amplification, and Characterization of Salmonella Isolates

Loop-mediated isothermal amplification (LAMP) assay was effective in detecting Salmonella enterica in naturally contaminated liquid egg samples. Salmonella was detected in 110 samples taken from four egg-breaking plants. The egg samples were pre-enriched in buffered peptone water (BPW) at 37°C for 20 h. The selective enrichment was done in Rappaport-Vassiliadis or tetrathionate broth and plated onto xylose lysine deoxycholate agar and brilliant green agar, modified. In addition, the PCR assay was used to detect Salmonella after pre-enrichment in BPW at 37°C for 20 h. The culture method and PCR assay were compared to the LAMP assay, which was also performed after pre-enrichment in BPW. PCR failed to detect Salmonella in 10% of 110 samples, whereas the culture method and LAMP assay successfully identified Salmonella in all samples. However, the LAMP assay was found to be much more rapid than the culture method and as sensitive in detecting Salmonella from liquid eggs. In all of the egg-breaking plants studied, Salmonella was isolated on most tested days. The positive samples showed that more than 75% of the Salmonella strains had identical genetic patterns when analyzed by pulsed-field gel electrophoresis. This suggests that the same Salmonella strains having survived long periods of time in the plants were contaminating the production line. The LAMP assay is rapid, specific, and sensitive for Salmonella detection in liquid eggs and is able to monitor Salmonella contamination in egg-handling plants more reliably.     

A NEW RAPID TECHNIQUE FOR SALMONELLA ANTIGEN DETECTION USING A PARTICLE CONCENTRATION FLUORESCENCE IMMUNOASSAY (PCFIA)

A fluorescent assay employing polystyrene beads coated with antibody to a common structural antigen (CSA) of salmonellae has been developed to detect the presence of Salmonella. This sensitive technique is very fast, relatively simple and with further testing may be suitable for screening food products as well as clinical diagnostics. Comparison was made with a commercially available Salmonella antigen capture enzyme immunoassay (EIA). As little as one single colony-forming unit (CFU) could be detected by fluorescence after only 16 h of enrichment. the procedure requires only 1 h to run and uses proportionately smaller volumes of sample and reagents than EIA.     

Poor performance of serological tests in the diagnosis of pulmonary tuberculosis: evidence from a contact tracing field study

Background

Delayed or missed diagnosis of TB continues to fuel the global TB epidemic, especially in resource limited settings. Use of serology for the diagnosis of tuberculosis, commonly used in India, is another factor. In the present study a commercially available serodiagnostic assay was assessed for its diagnostic value in combination with smear, culture and clinical manifestations.

Methodology/Principal Findings

A total of 2300 subjects were recruited for the study, but 1041 subjects were excluded for various reasons. Thus 1259 subjects were included in the study of which 470 were pulmonary tuberculosis cases (440 of 470 were culture-positive) and 789 were their asymptomatic contacts. A house-to-house survey method was used. Blood samples were tested for IgM, IgA, and IgG antibodies using the Pathozyme Myco M (IgM), Myco A (IgA) and Myco G (IgG) enzyme immunoassay (EIA). Out of 470 PTB cases, BCG scar was positive in 82.34%. The Mantoux test and smear positivity rates in PTB cases were 94.3% (430/456), and 65.32% (307/470), respectively. Among the asymptomatic contacts, BCG scar was positive in 95.3% and Mantoux test was positive in 80.66% (442/548) contacts. No contact was found falsely smear positive. The sensitivity of IgM, IgA, and IgG EIA tests was 48.7%, 25.7% and 24.4%, respectively, while the specificity was 71.5%, 80.5%, 76.6%, respectively. Performance of EIAs was not affected by the previous BCG vaccination. However, prior BCG vaccination was statistically significantly (p = 0.005) associated with Mantoux test positivity in PTB cases but not in contacts (p = 0.127). The agreement between serology and Mantoux test was not significant.

Conclusion

The commercial serological test evaluated showed poor sensitivity and specificity and suggests no utility for detection of pulmonary tuberculosis.     

Gold nanoparticles-based colorimetric assay for rapid detection of Salmonella species in food samples

A rapid, sensitive and cost-effective method was developed for detection of foodborne pathogens, particularly Salmonella species. The method utilizes single stranded DNA (ssDNA) probes and non-functionalized gold nanoparticles to provide a colorimetric assay for the detection of PCR amplified DNA. Different food samples were tested with the PCR-based colorimetric assay parallel with the conventional culture method. The sensitivity and specificity of colorimetric assay was 89.15 and 99.04% respectively with reference to conventional culture method. The total time required to detect the Salmonella spp. present in food samples by the developed method is less than 8 h, including 6 h incubation. It was observed that the colorimetric assay was 10 times more sensitive than gel-based detection with the same concentration of DNA used for analysis.