Sandwich capture ELISA by a murine monoclonal antibody against a genus-specific LPS epitope for the detection of different common serotypes of salmonellas.

A sandwich capture ELISA based on a murine monoclonal antibody against a genus-specific epitope in the outer core region of the Salmonella lipopolysaccharide is described for the detection of different common serotypes of salmonellas. Four h broth cultures of seven standard and 24 wild strains of salmonellas were all detected by the capture ELISA while overnight broth cultures of 21 non-salmonella standard strains were all negative. The capture ELISA detected 1 ng/ml of Ra lipopolysaccharide, 10(6)/ml of a smooth wild strain of Salm. typhimurium, and 1120 cells of Salm. heidelberg after enrichment culture for 4 h.     

Diagnostic application of monoclonal antibodies to outer membrane protein for rapid detection of Salmonella.

Monoclonal antibodies produced to Salmonella enteritidis outer membrane proteins were screened against 57 Salmonella serovars and several related enterobacteria. Those detecting all Salmonella serovars and none of the related enterobacteria were used in a microtitre plate antigen capture ELISA to screen clinical samples. Sixty-one of 2100 samples yielded salmonellas after incubation for 24 h in selective media by conventional culture. Of these 58 were detected by the ELISA. Sixty-five false positives by ELISA were found to be Enterobacter spp. The results show the potential of this ELISA to eliminate a large proportion of the salmonella-negative cultures at an early stage.     

Sandwich capture ELISA by a murine monoclonal antibody against a genus-specific LPS epitope for the detection of different common serotypes of salmonellas

D.CHOI, R.S.W. TSANG AND M.H. NG. 1992. A sandwich capture ELISA based on a murine monoclonal antibody against a genus-specific epitope in the outer core region of the Salmonella lipopolysaccharide is described for the detection of different common serotypes of salmonellas. Four h broth cultures of seven standard and 24 wild strains of salmonellas were all detected by the capture ELISA while overnight broth cultures of 21 non-salmonella standard strains were all negative. The capture ELISA detected 1 ng/ml of Ra lipopolysaccharide, 10⁶/ml of a smooth wild strain of Salm. typhimurium , and 1120 cells of Salm. heidelberg after enrichment culture for 4 h.     

Evaluation of an enzyme immunoassay technique for the detection of salmonellas in minced meat

A comparison was made between a commercially available enzyme immunoassay (ELISA) and various cultural procedures for detecting salmonellas in minced meat contaminated with a standard inoculum. To detect salmonellas by the ELISA technique, it was necessary to modify the recommended baseline for spectrophotometric measurements to avoid false-positive results. The incidence of false-negatives was no greater than that obtained with a standard isolation procedure. Both methods were affected by the competing microflora.     

An improved ELISA method for the detection of Salmonella typhimurium

The applicability of enzyme-linked immunosorbent assay (ELISA) for the detection of salmonellas in foodstuffs was investigated. Several factors affecting the sensitivity of the ELISA, such as the type of protein used for plate post-coating, the method of antibody labelling, and accelerators for antigen-antibody and enzyme-substrate reactions, were studied. Labelling of the antibody with horseradish peroxidase and the use of o -phenylenediamine as substrate in the detection system were demonstrated to be most suitable for the enzyme assay.
Based on these findings, an improved ELISA method was developed for the detection of Salmonella typhimurium. The improved technique was able to detect as few as 5×10⁴-10⁵ cell/ml of salmonellas, and about 24 h were required to enrich the bacteria in food samples and to perform the test. With some modifications, the ELISA assay could reach a very high level of sensitivity and provide excellent repro-ducibility.     

An improved ELISA method for the detection of Salmonella typhimurium

The applicability of enzyme-linked immunosorbent assay (ELISA) for the detection of salmonellas in foodstuffs was investigated. Several factors affecting the sensitivity of the ELISA, such as the type of protein used for plate post-coating, the method of antibody labelling, and accelerators for antigen-antibody and enzyme-substrate reactions, were studied. Labelling of the antibody with horseradish peroxidase and the use of o-phenylenediamine as substrate in the detection system were demonstrated to be most suitable for the enzyme assay. Based on these findings, an improved ELISA method was developed for the detection of Salmonella typhimurium. The improved technique was able to detect as few as 5 x 10(4)-10(5) cell/ml of salmonellas, and about 24 h were required to enrich the bacteria in food samples and to perform the test. With some modifications, the ELISA assay could reach a very high level of sensitivity and provide excellent reproducibility.     

Diagnostic application of monoclonal antibodies to outer membrane protein for rapid detection of salmonella

S. KERR. H.J. BALL, D.P. MACKIE, D.A. POLLOCK AND D.A. FINLAY. 1992. Monoclonal antibodies produced to Salmonella enteritidis outer membrane proteins were screened against 57 Salmonella serovars and several related enterobacteria. Those detecting all Salmonella serovars and none of the related enterobacteria were used in a microtitre plate antigen capture ELISA to screen clinical samples. Sixty-one of 2100 samples yielded salmonellas after incubation for 24 h in selective media by conventional culture. Of these 58 were detected by the ELISA. Sixty-five false positives by ELISA were found to be Enterobacter spp. The results show the potential of this ELISA to eliminate a large proportion of the salmonella-negative cultures at an early stage.     

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.     

Dynamics of salmonella isolation with modified Rappaport's medium (R10)

Enhanced growth of salmonellas in Rappaport's medium as modified by Vassiliadis et al. (1976) after pre-enrichment in buffered peptone water during the first 6 h was obtained by replacement of tryptone by soya peptone. The competing bacteria, i.e. those which grow on brilliant green agar and which may interfere with the isolation of salmonellas when Rappaport's medium (R10) incubated at 43°C is used for enrichment were inhibited or reduced in numbers when the normal amount of 5 g soya peptone/litre was used. When the amount was increased to 10 g/l, growth occurred, mainly of Enterobacter and Klebsiella species. The isolation of salmonellas was found to be largely dependent on the number and the ratio of their competitors. Every measure taken to reduce the number of competitive bacteria increases the possibility of isolating salmonellas. This explains the effect of improved selectivity of Rappaport's medium when small inocula are used. Rapid onset of growth of salmonellas by employing soya peptone introduces the possibility of using shorter incubation times than 48 h.     

Comparison of ELISA with shell vial cell culture method for the detection of human rotavirus in fecal specimens

The aim of the study was to compare an enzyme immunoassay method with shell vial cell culture method for detection of rotavirus in fecal specimens. In addition, the correlation between laboratory results and clinical scores of patients with gastroenteritis was evaluated. A total of 219 fecal specimens from children (ages 3 weeks to 5 years) with acute gastroenteritis submitted to pediatric emergency room were evaluated by both ELISA and shell vial cell culture. A Vesikari score was used for assessing the severity of the illness. Among 219 stool samples tested, 107 (48.9%) were determined to be positive. Two specimens were positive by shell vial cell culture method while they were ELISA negative. According to these results the calculated sensitivity, specificity, PPV, and NPV of ELISA were 98.1%, 100%, 100%, and 98.2%, respectively. The mean severity score for the 107 episodes of rotavirus diarrhoea was 11.0 +/- 3.6 compared to 4.5 +/- 1.9 for the 112 episodes of non-rotavirus diarrhea in the same population. Our study indicates that ELISA, which is easier to perform, faster and cheaper than cell culture methods may be suitable for routine diagnosis of rotavirus infections. The severity of rotavirus positive gastroenteritis was significantly higher than that of rotavirus negative patients.     

Dynamics of salmonella isolation with modified Rappaport's medium (R10)

Enhanced growth of salmonellas in Rappaport's medium as modified by Vassiliadis et al. (1976) after pre-enrichment in buffered peptone water during the first 6 h was obtained by replacement of tryptone by soya peptone. The competing bacteria, i.e. those which grow on brilliant green agar and which may interfere with the isolation of salmonellas when Rappaport's medium (R10) incubated at 43 degrees C is used for enrichment were inhibited or reduced in numbers when the normal amount of 5 g soya peptone/litre was used. When the amount was increased to 10 g/l, growth occurred, mainly of Enterobacter and Klebsiella species. The isolation of salmonellas was found to be largely dependent on the number and the ratio of their competitors. Every measure taken to reduce the number of competitive bacteria increases the possibility of isolating salmonellas. This explains the effect of improved selectivity of Rappaport's medium when small inocula are used. Rapid onset of growth of salmonellas by employing soya peptone introduces the possibility of using shorter incubation times 48 h.     

Viable but non-culturable salmonellas in soil

P.E. TURPIN, K.A. MAYCROFT, C.L. ROWLANDS AND E.M.H. WELLINGTON. 1993. An enzyme-linked immunosorbent assay (ELISA) and a microwell fluorescent antibody (FA) direct count method have been developed for the monitoring of salmonellas in soil. Both methods have a minimum detection level of ca 10⁶ cells per gram of soil. The FA direct count method gave a linear recovery for the inoculum range 10⁶–10⁹ cells per gram of soil. When monitored by plate counts the survival of salmonellas was greater in a sterile than in a non-sterile soil. Evidence was found for the production of viable but non-culturable salmonellas in non-sterile soil; plate counts dropped rapidly with time, but FA direct counts and ELISA remained level. The salmonella cells became progressively smaller and rounder with time. Dead salmonella cells introduced into soil rapidly disappeared.     

Development of a 24 h screen to detect viable salmonellas in faeces

A 24 h screen to detect viable salmonellas in faeces was developed by studying growth dynamics of salmonellas and competing flora in combinations of enrichment media and artificially-inoculated pig faeces. Muller-Kauffmann tetrathionate (MK) broth, incubated overnight at 42°C, maintained the lowest ratio of salmonella: competing flora and identified all inoculated samples. A 4 h postenrichment in M broth plus novobiocin reduced the number of false-positive results in subsequent ELISAs. Adjusting the negative cut-off values and incubation time of the chromogenic substrate from that recommended in the ELISA instructions reduced the rate of false-positive results further and allowed the detection of 10³ salmonellas per ml in the presence of up to 10⁷ ml⁻¹ aerobic-competing cells. Suspension of faeces diluted 1 in 2 and 1 in 5, rather than 1 in 10 in MK broth did not necessitate further adjustments to the ELISA baseline values. The proposed screen protocol is an overnight incubation of faeces suspended 1 in 10 in MK broth, a 1 in 100 subculture into M broth plus 10 μg ml⁻¹ novobiocin (MbN) for 4 h, steam inactivation of MbN cultures and testing by ELISA, and can detect three salmonella cells per g faeces.     

Effect of carbohydrate source in selenite cystine trimethylamine oxide broth on the detection of salmonellas using the Bactometer

Ninety-five salmonellas and 40 non-salmonellas were screened in the Bactometer using the standard formulation for Easter and Gibson's selenite cystine trimethylamine oxide dulcitol broth and versions in which dulcitol was replaced by mannitol or deoxyribose. More strains of salmonellas exceeded the current detection criteria (magnitude 250, rate 25) when dulcitol was replaced by either mannitol or deoxyribose as carbohydrate source. Using mannitol, more non-salmonella strains exceeded the detection criteria than with either dulcitol or deoxyribose.     

Fluorescent detection-polymerase chain reaction (FD-PCR) assay on microwell plates as a screening test for salmonellas in foods

This study evaluates a polymerase chain reaction assay coupled with a fluorescent detection in microwell plates for salmonellas in food samples. Chelex 100-extracted cultures and bulk and processed food samples were used as templates for a PCR assay in microwell plates, with a primer pair that amplifies a 206 bp segment of IS 200 . The PCR products were then denatured by heat and transferred to CovaLink NH plates (Nunc) to which capture oligonucleotides were covalently bound. Hybridization was performed for 1 h at 55°C, the microwells were washed and an alkaline phosphatase-labelled probe, complementary of an internal sequence of the PCR product, was added. After stringent washes, 100 μl of 1 mmol 1^-1 AttoPhos^TM (JBL Scientific) was then added to the wells and the fluorescence measurement system (Millipore). The level of detection of the assay was as low as 1–10 cfu. A total of 172 food samples were tested, both by culture and FD-PCR. Of these 53 were culture positive and 119 culture negative. The sensitivity of the FD-PCR assay was 100% and the specificity was 90.1%. Positive and negative predictive values were 82.8 and 100%, respectively. Based on the results obtained in this study it appears that the FD-PCR. assay described here can be useful to screen a large number of food samples for contamination by salmonellas.     

Inactivation of Salmonella during anaerobic digestion of sewage sludge

The inactivation of Salmonella duesseldorf in sewage sludge during anaerobic digestion was investigated at 35 and 48°C with mean retention periods of between 10 and 20 days. Digesters were fed daily with raw sludge containing added Salm. duesseldorf after removal of digested sludge. During steady operation, the levels of Salm. duesseldorf in the digested and the feed sludge were determined and their specific rates of decay were estimated. The latter were: (i) greater at 48°C than at 35°C for the same retention time; (ii) similar for retention periods greater than 15 d, but lower for 10 d; (iii) greater when the level of salmonellas in the feed was lower. Gas production, a measure of steady state, was gradually lost when the mean retention period was reduced to 6.7 d. In experiments in which a single dose of Salm. duesseldorf was added to digesting sludge, the inactivation appeared to follow first-order kinetics at 35°C and the decimal decay rate, 1.6/d, was similar to that in the daily feeding experiments (1.4/d) with larger and similar inocula of Salm. duesseldorf. At 48°C, however, the rate of inactivation declined with decreasing time from inoculation suggesting that the culture contained cells differing in thermal resistance. The degrees and rates of inactivation of salmonellas in those experiments were greater than in full-scale digesters, because the latter seldom operated under conditions ideal for inactivation or because indigenous salmonellas are more resistant.     

Enzyme-linked immunosorbent assay for detection of Salmonella lipopolysaccharide in poultry specimens.

An enzyme-linked immunosorbent assay (ELISA) for detection of salmonellae was developed and evaluated by using artificially contaminated specimens of poultry feed, feces, litter, or carcass rinsings, and naturally contaminated water samples. Specimens containing salmonellae of serogroups B or C2 inhibited the binding of polyvalent anti-O serum to microtiter plate wells coated with lipopolysaccharide of Salmonella typhimurium (serogroup B) or Salmonella albany (serogroup C2), respectively. Treatment of specimens with Rhozyme 41 (a protease) inhibited nonspecific reactions. The ELISA detected 106 of 111 culture-positive specimens contaminated with salmonellae of serogroups B or C2. Nineteen of 20 specimens containing salmonellae of serogroup C1 and all of 36 culture-negative specimens were ELISA negative. All seven water samples that contained salmonellae of serogroups B or C2, including three that were culture positive only after delayed secondary enrichment, were ELISA positive. Seven of the nine water samples that contained salmonellae of other serogroups, and all 38 culture-negative samples, were ELISA negative. The ELISA was simple to perform, produced results in 48 h, and was more economical than culture methods.     

Enzyme-linked immunosorbent assay for detection of Salmonella lipopolysaccharide in poultry specimens

An enzyme-linked immunosorbent assay (ELISA) for detection of salmonellae was developed and evaluated by using artificially contaminated specimens of poultry feed, feces, litter, or carcass rinsings, and naturally contaminated water samples. Specimens containing salmonellae of serogroups B or C2 inhibited the binding of polyvalent anti-O serum to microtiter plate wells coated with lipopolysaccharide of Salmonella typhimurium (serogroup B) or Salmonella albany (serogroup C2), respectively. Treatment of specimens with Rhozyme 41 (a protease) inhibited nonspecific reactions. The ELISA detected 106 of 111 culture-positive specimens contaminated with salmonellae of serogroups B or C2. Nineteen of 20 specimens containing salmonellae of serogroup C1 and all of 36 culture-negative specimens were ELISA negative. All seven water samples that contained salmonellae of serogroups B or C2, including three that were culture positive only after delayed secondary enrichment, were ELISA positive. Seven of the nine water samples that contained salmonellae of other serogroups, and all 38 culture-negative samples, were ELISA negative. The ELISA was simple to perform, produced results in 48 h, and was more economical than culture methods.     

Malachite green pre-enrichment medium for improved salmonella isolation from heavily contaminated samples

Large numbers of competitive bacteria may hinder the isolation of salmonellas from food and environmental samples when a pre-enrichment method is used. The addition of 0.1 g/l of malachite green (MG) to buffered peptone water (BPW) inhibited the multiplication of Gram-positive bacteria. Brilliant green had a similar effect but only when the normal recommended concentration of 0.02 g/l was raised to 0.05 g/l. Pure strains of salmonellas were inhibited by MG in BPW, but addition of non fat dried milk (NFDM) (5 g/l or more) counteracted this effect. MG did not affect the recovery of salmonellas injured by heat, freezing, low water activity or acidity in BPW with NFDM. It was concluded that addition of MG to BPW may improve the possibility of isolating salmonellas from heavily contaminated materials by limiting the competitive growth of Gram-positive bacteria and the subsequent lowering of the pH of the broth.     

用改进的ELISA法检测腺病毒抗原

用改进的ELISA法检测94份咽拭子标本中的腺病毒抗原,标本先接种人肾细胞,使病毒有一定程度的增殖,同时与传统的病毒分离作对比,结果表明,腺病毒分离阳性的18例,ELISA法检测全部阳性,76例分离阴性(盲传3代)者ELISA法也全都阴性,两者完全相符,ELISA法检测增殖24、48、72小时的细胞培养物腺病毒抗原的阳性率分别为16.7％,72.2％和83.3％,本法特异、可靠、判断客观、可用来检测腺病毒抗原。