Evaluation of Frozen Fixed Smears for Use in Fluorescent Antibody Studies of Salmonellae

Smears of broth cultures of 28 Salmonella serotypes were fixed with Kirkpatrick fixative and stored at -20 C. Results indicate that organisms retain the ability to stain at maximal fluorescence intensity for as long as 2 years.     

Comparison of Enrichment Procedures for Fluorescent Antibody and Cultural Detection of Salmonellae in Raw Meat and Poultry

No advantage was shown in preenriching raw meat samples for detecting salmonellae by fluorescent antibodies or culture. Trypticase soy-tryptose (Edwards and Ewing, 1972) was equal to or better than selenite-cystine as a postenrichment broth.     

Development and Evaluation of a Direct Fluorescent Antibody Method for the Diagnosis of Pneumocystis carinii Infections in Experimental Animals

Because no fully satisfactory diagnostic method has been available for use in pneumocystis infection, an attempt was made to apply the fluorescent antibody technique in the identification of Pneumocystis carinii. Hyperimmune sera were prepared in rabbits against P. carinii from human and rat sources. After proper adsorption, these antisera were conjugated with fluorescein isothiocyanate and used as reagents in a direct fluorescent antibody procedure. Each of the two reagents was found to stain trypsin-treated P. carinii organisms from either human or rat sources, indicating the presence of common antigens. Stained organisms were demonstrated in the hypopharyngeal material from rats in which pneumocystis infection had been activated by the administration of corticosteroid. From the results reported here, the procedure outlined is considered sufficiently sensitive and specific to justify tests on pneumocystis infections in man. The findings in a series of specimens from human subjects will be reported separately. The method also provides an extended approach to related research problems. The need for controls of the procedure at all points is emphasized.     

Rapid Detection of Salmonella Microcolonies by Fluorescent Antibody

A microcolony fluorescent-antibody (FA) procedure for detecting salmonellae was compared to the usual direct FA procedure on 304 environmental, food, and feed samples. The microcolony FA test detected all of the specimens found positive by culture, whereas the direct FA missed 3.1% of them. Both FA tests revealed stained organisms in some of the culturally negative specimens. The microcolony FA test has several advantages over the direct FA test: ease of examining the smears, elimination of the fluorescent background material, and increased sensitivity.     

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.     

Detection of Air-borne Pasteurella tularensis Using the Fluorescent Antibody Technique

Considerable research has been directed toward the development of rapid methods for the identification of air-borne microorganisms. The application of the fluorescent antibody technique (FAT) coupled with the impaction of contaminated air onto glass slides affords a rapid and specific method for the identification of air-borne Pasteurella tularensis. Early experiments presented problems of cross-reaction with organisms other than P. tularensis. These cross-reactions are eliminated by specific adsorption and proper dilution of the conjugate. A series of experiments conducted under rigidly controlled laboratory conditions indicates that fewer than ten viable P. tularensis per slide can be detected by this method. Time of impaction as well as the presence of large concentrations of other microorganisms did not alter this number. Calculations indicate that a concentration as low as one viable organism per 5 liters of air can be detected.     

Automated Fluorescent Treponemal Antibody Test: Instrument and Evaluation

Two evaluations of the automated fluorescent treponemal antibody (AFTA) test for the serodiagnosis of syphilis are described. The results of AFTA and manually performed fluorescent treponemal antibody-absorption (FTA-ABS) tests were compared on serum samples from clinically defined donor groups, and the reproducibility of each procedure was studied. Significant improvement of AFTA test results was obtained in the most recent study after developmental modifications of the instrument and test technique. AFTA test agreement with both syphilis and nonsyphilis categories was considered good. With the increasing usage of the FTA-ABS test as an effective tool for the diagnosis of syphilis, successful automation of this procedure is particularly timely and significant.     

Evaluation of Commercial Fluorescein Isothiocyanates Used in Fluorescent Antibody Studies

Commercial preparations of fluorescein isothiocyanate (FITC) for immunofluorescence applications were obtained from 12 sources and examined for purity by quantitative infrared spectrophotometry and by labeling efficiency for bovine serum albumin (BSA). Quantitative photometric measurements were made of nonspecific staining (NSS) produced by conjugates prepared from the dyes. The purity of FITC from different sources was highly variable. The risk of NSS appears to increase as the purity of the dye decreases. In immunofluorescence applications it is desirable to use the purest FITC available in order to obtain conjugates with minimum NSS. It is recommended that 70% FITC, as determined by BSA labeling efficiency, be accepted as the minimum purity for immunofluorescence applications.     

A Comparison of the Fluorescent Antibody Method and a Standardized Cultural Method for the Detection of Salmonellas

The results of routine use of the indirect fluorescent antibody (FA) technique using the Spicer-Edwards H antisera set are reported for a range of agricultural and food samples. The FA technique was used on samples after the pre-enrichment incubation period in the proposed ISO method for isolation of salmonellas. The numbers of FA false positive samples ( ca. 5% overall) and FA false negative samples ( ca. 1·3%) were low, but some originally FA false positive results were later shown to be false negative cultural results.     

Detection of Salmonella in Eggs and Egg Products With Fluorescent Antibody

Organisms of the genus Salmonella are detected in eggs and egg products within 24 hr in the presence of Pseudomonadaceae and other Enterobacteriaceae by combining selective cultural methods with fluorescent-antibody techniques. These techniques are specific for Salmonella when H antibodies are used. Absorption techniques are necessary before the O antibodies give specific reactions for Salmonella. No cross-reactions appear when H antiserum is used. Absorption and interference techniques indicate the test is specific for Salmonella.     

Detection of Salmonellae in the Environment

The incidence of salmonellae in contrasting environments was compared in this study. Samples collected from or near surface waters in a lush hardwood forest yielded four salmonellae serotypes from six culturally positive samples. A total of 76 samples collected from the top of a granite outcropping over a 3-month period yielded 10 positive samples. Only two salmonellae serotypes were isolated, and one of these was isolated only once. The nature of the sample material had no significant effect on the detection of salmonellae from the two sampling sites. However, the presence or absence of visible moisture in the sample significantly affected the recovery of salmonellae. The results showed that even a harsh environment such as that found on top of Stone Mountain may serve as an ecological niche for the survival and transmission of salmonellae.     

Evaluation of the Salmonellae Fluoro-Kit for Fluorescent-Antibody Staining

An evaluation of the newly developed Clinical Sciences, Inc. Salmonellae Fluoro-Kit, which attempts to standardize the various aspects of the fluorescent-antibody (FA) procedure, was performed with 120 naturally contaminated human food, animal feed, and raw material samples. The Association of Official Analytical Chemists (AOAC) method for the detection of salmonellae was used as the control method. The Fluoro-Kit was found to be simple and conveniento to use. The results of this preliminary study show an industrially acceptable rate of recovery of salmonellae by using the Fluoro-Kit in comparison with the A.O.A.C. method. The Fluoro-Kit shows promise as a rapid, salmonellae FA screening method. Problems originally encountered in the application of the Fluoro-Kit are discussed. According to the manufacturer, strict adherence to the now revised procedures included in the Fluoro-Kit will control these problems.     

Evaluation of the Interference Filter for Use in Rabies Diagnosis by the Fluorescent Antibody Test

When compared with primary filters widely used for rabies diagnosis by the fluorescent antibody test, an interference filter markedly increased specific staining intensity and contrast.     

A Novel Chromogenic Ester Agar Medium for Detection of Salmonellae

A novel agar medium, chromogenic Salmonella esterase (CSE) agar, for the differentiation of salmonellae is described. The agar contains peptones and nutrient extracts together with the following (grams per liter unless otherwise specified): 4-[2-(4-octanoyloxy-3,5-dimethoxyphenyl)-vinyl]-quinolinium-1-(propan-3-yl carboxylic acid) bromide (SLPA-octanoate; bromide form), 0.3223; lactose, 14.65; trisodium citrate dihydrate, 0.5; Tween 20, 3.0; ethyl 4-dimethylaminobenzoate, 0.035% (wt/vol), novobiocin, 70 mg liter⁻¹. The key component of the medium is SLPA-octanoate, a newly synthesized ester formed from a C₈ fatty acid and a phenolic chromophore. In CSE agar, the ester is hydrolyzed by Salmonella spp. to yield a brightly colored phenol which remains tightly bound within colonies. After 24 h of incubation at 37 or 42°C, colonies of typical Salmonella spp. were burgundy colored on a transparent yellow background, whereas non-Salmonella spp. were white, cream, yellow or transparent. CSE agar was evaluated by using a panel of strains including a high proportion of Salmonella and non-Salmonella strains giving atypical reactions on other differential agars. The sensitivity (93.1%) of CSE agar for non-typhi salmonellae compared favorably with those of Rambach (82.8%), xylose-lysine-deoxycholate (XLD; 91.4%), Hektoen-enteric (89.7%), and SM ID (91.4%) agars. The specificity (93.9%) was also comparable to those of other Salmonella media (SM ID agar, 95.9%; Rambach agar, 91.8%; XLD agar, 91.8%; Hektoen-enteric agar, 87.8%). Strains of Citrobacter freundii and Proteus spp. giving false-positive reactions with other media gave a negative color reaction on CSE agar. CSE agar enabled the detection of >30 Salmonella serotypes, including agona, anatum, enteritidis, hadar, heidelberg, infantis, montevideo, thompson, typhimurium, and virchow, which accounted for 91.8% of the salmonella isolates recorded by the Public Health Laboratory Service (Colindale, London, England) for 1997.     

Evaluation of a DNA probe test kit for detection of Salmonellae in biosolids

AIMS: Current US regulations (40 CFR 503) for 'Class A' biosolids (treated sewage sludge) requires use of multiple-tube fermentation techniques for fecal coliform or multiple tube enrichment techniques for Salmonella spp. followed by isolation and biochemical and serologic confirmation. The technical difficulties and the time required to complete the procedure for enumeration of Salmonellae in biosolids and sludges has limited the use of this assay. This study was conducted to determine if a commercially available molecular probe system could be used to isolate and enumerate Salmonella spp. in biosolids or sludges in less time than cultural techniques with biochemical confirmation. METHODS AND RESULTS: Several types of treated and untreated municipal sludges were assayed for Salmonellae using a cultural technique with biochemical and serologic confirmation and a DNA probe diagnostic test kit. The results indicate that the molecular probe and the conventional fermentation tube technique yielded equivalent results. Interestingly, the probe technique yielded results within 52 h following initiation of sample analysis compared with the conventional fermentation tube technique with confirmation which required approx. 120 h. CONCLUSIONS: These results suggest that the molecular probe system used for this work may be used to determine the presence or absence of Salmonella spp. in biosolids within a relatively short time frame. SIGNIFICANCE AND IMPACT OF THE STUDY: The ease of using the DNA probe test kit, along with its ability to produce results in less than half the time of conventional culture techniques, suggests that this assay is useful for determining the presence or absence of Salmonellae in biosolids samples.     

Preparation and Testing of Polyvalent Conjugates for Fluorescent-Antibody Detection of Salmonellae

A polyvalent OH conjugate for Salmonella O groups A through I, K, L, and O was prepared and tested against pure cultures of salmonellae, nonsalmonellae, and a variety of food, fecal, and environmental specimens. Examination of pure cultures revealed that the conjugate gave negligible staining with representative strains of Shigella, Proteus, Providence, Serratia, and Pseudomonas. However, it stained 12% of the Escherichia coli and Citrobacter freundii strains and 36% of the Arizona strains. Over 1,200 specimens of various types were examined by both fluorescent-antibody (FA) and cultural procedures. Results indicate that, when used with discretion, FA screening can be a useful tool for rapid presumptive indication of the presence of salmonellae. The need for careful selection of strains used for preparing antisera and the importance of adequate evaluation of Salmonella FA reagents are discussed.     

Direct,Specific and Rapid Detection of Staphylococcal Proteins and Exotoxins Using a Multiplex Antibody Microarray

Background

S. aureus is a pathogen in humans and animals that harbors a wide variety of virulence factors and resistance genes. This bacterium can cause a wide range of mild to life-threatening diseases. In the latter case, fast diagnostic procedures are important. In routine diagnostic laboratories, several genotypic and phenotypic methods are available to identify S. aureus strains and determine their resistances. However, there is a demand for multiplex routine diagnostic tests to directly detect staphylococcal toxins and proteins.

Methods

In this study, an antibody microarray based assay was established and validated for the rapid detection of staphylococcal markers and exotoxins. The following targets were included: staphylococcal protein A, penicillin binding protein 2a, alpha- and beta-hemolysins, Panton Valentine leukocidin, toxic shock syndrome toxin, enterotoxins A and B as well as staphylokinase. All were detected simultaneously within a single experiment, starting from a clonal culture on standard media. The detection of bound proteins was performed using a new fluorescence reading device for microarrays.

Results

110 reference strains and clinical isolates were analyzed using this assay, with a DNA microarray for genotypic characterization performed in parallel. The results showed a general high concordance of genotypic and phenotypic data. However, genotypic analysis found the hla gene present in all S. aureus isolates but its expression under given conditions depended on the clonal complex affiliation of the actual isolate.

Conclusions

The multiplex antibody assay described herein allowed a rapid and reliable detection of clinically relevant staphylococcal toxins as well as resistance- and species-specific markers.     

Further Evaluation of a Rapid Immunofluorescence Technique for Detecting Salmonellae in Meat and Poultry

A rapid 18–24 h immunofluorescence technique detected 14 of 15 positive samples in tests on 706 routine samples, which included 656 home produced raw beef samples. The rapid technique also recorded 49 false positive results, i.e. samples which proved negative in subsequent cultural tests. The immunofluorescence technique could be used as a presumptive screening test aimed at the rapid detection of negative samples. In this way salmonella free raw materials should usually be cleared for production within 1 day of sampling.     

Direct Addition of Liquid Propylene Oxide to Dried Materials for Destruction of Salmonellae

Propylene oxide added in liquid form to dried materials (e.g., animal by-products) is highly effective for destroying salmonellae.     

Newly Established Monoclonal Antibody Diagnostic Assays for Schistosoma mansoni Direct Detection in Areas of Low Endemicity

Background

Current available methods for diagnosis of schistosomiasis mansoni lack sufficient sensitivity, which results in underreporting of infectious in areas of low endemicity.

Methodology/Principal Findings

We developed three novel diagnostic methodologies for the direct detection of schistosome infection in serum samples. These three new methods were evaluated with positive patients from a low endemicity area in southeast Brazil. The basis of the assay was the production of monoclonal antibodies against the protein backbone of heavily glycosylated Circulating Cathodic Antigen (CCA). The antibodies were also selected for having no specificity to repeating poly-Lewis x units. Assays based on the detection CCA-protein should not encounter a limitation in sensitivity due to a biological background of this particular epitope. Three diagnostic methodologies were developed and validated, (i) Immunomagnetic Separation based on improved incubation steps of non-diluted serum, (ii) Direct Enzyme-linked Immunosorbent Assay and (iii) Fluorescent Microscopy Analysis as a qualitative assay. The two quantitative assays presented high sensitivity (94% and 92%, respectively) and specificity (100%), equivalent to the analysis of 3 stool samples and 16 slides by Kato-Katz, showing promising results on the determination of cure.

Conclusions/Significance

The Immunomagnetic Separation technique showed excellent correlation with parasite burden by Cohen coefficient. The qualitative method detected 47 positive individuals out of 50 with the analysis of 3 slides. This easy-to-do method was capable of discriminating positive from negative cases, even for patients with low parasite burden.