William B. Cherry

DETECTION OF SALMONELLAE IN FOODSTUFFS, FECES, AND WATER BY IMMUNOFLUORESCENCE

The major impetus for development of polyvalent Salmonella immunofluorescent ( IF) reagents has come from the food industries and from the regulatory agencies charged with insuring a safe food supply. Among the gram-negative bacteria, the salmonellae probably produce more infections in more species of animals in more geographic areas of the world than any other organism. In the United States alone, it has been estimated that reported isolations of Salmonella account for only about 1% of the actual human cases.j5 Because approximately 25,000 isolates are reported annually, the morbidity may easily reach 2,500,000 cases.36 Protection against infection by the salmonellae encompasses the provision of pure food and water and interruption of the direct and indirect fecal-oral transmission routes. If consumers are t o be protected from infection, mass-produced foods, such as meat, fish, dairy products, and processed foods, derived from these sources must be monitored for the presence of these ubiquitous organisms. The cost of conventional cultural procedures for isolation and identification of the salmonellae was estimated in 1971 by the U.S. Department of Agriculture (USDA) and by the Food and Drug Administration (FDA) t o be about 5-7 dollars per sample. The use of I F tests for mass screening of foodstuffs and water offers an economic alternative to conventional procedures.

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.

Report on a Study Set of Moraxellae and Allied Bacteria

Sixty-two strains of moraxellae and allied bacteria assembled by the Subcommittee on Moraxella and Allied Bacteria of the International Committee on Systematic Bacteriology and distributed by the American Type Culture Collection were examined serologically by the fluorescent-antibody (FA) and quellung techniques and subjected to study using 71 biochemical tests. These strains, originally assigned to 11 different genera, were placed in two categories: Acinetobacter and Moraxella. There were 41 strains assigned to Acinetobacter and 19 to Moraxella. Two strains were eliminated from the study. One strain belonged to neither of these genera; the other was eliminated on the basis of colonial and biochemical variability. Sixty per cent of the carbohydrate-oxidizing strains of Acinetobacter were typable by FA and quellung reaction in conjugates and sera prepared from oxidizing strains. The capsular antigens of the oxidizers (formerly called Herellea) are distinctly different from those of the nonoxidizers (formerly called Mima). A small percentage of the nonoxidizing cultures exhibited cross-reactions with antibodies for the oxidizers. None of the moraxellae were typable with conjugates or antisera for the oxidizing acinetobacters. The results of these studies were correlated with those of other workers. In general, the classification of the bacteria discussed in this report was quite reliable when based on the use of seven selected phenotypic characteristics. This fact has practical diagnostic importance. The true relationships and taxonomic placements of the moraxellae and allied bacteria are dependent upon the development and employment of systems for genetic analysis. On the basis of studies reviewed in this paper, most workers would now agree upon the separation of the oxidase-positive and oxidase-negative organisms with assignment of the former to the genus Moraxella Lwoff and of the latter to the genus Acinetobacter Brisou and Prevot.

Optimum immunization of rabbits for Streptococcus mutans antiserum and conjugate production and studies of batch immunoabsorption methods.

By far, the most significant rises in titers were seen with the immunization protocol used in series 6. Conjugates prepared from bleedings on the 33rd day produced exceptionally high titers for type b S mutans, and reasonably high titers for type a were obtained in a short time. A concentrated antigen with Formalin (13.4 ml) was given during a ten-day period followed by a two-week rest period, after which booster doses of either antigen with Formalin or live antigen were given (Fig 1). Based on evaluation of the immunization protocol just described, series 6 resulted in the highest titered reagents, but the data are insufficient to permit recommending that particular schedule without limitations. Our experience in the use of live antigens of S mutans for immunization is limited in that only types b, c, and e have been used in this way. The rabbits survived these injections, but the pathogenicity of other strains and other serotypes has not been determined. In addition, protocols including combined injections of killed and living organisms should be tested further for possible improvement in antibody production. In view of these considerations, our recommendations for production of high titered antiserums for S mutans in rabbits are as follows: -Take a preimmunization bleeding from each rabbit and screen by indirect FA tests with the antigens to be used. -Inject heavy concentrations (40 IU/ml) of Formalin-killed cells, intravenously. -Inject for eight to ten consecutive days, giving increasing doses of antigen ranging from 0.2 to 5.0 ml for a total of 12 to 15 ml. -Rest the rabbits for one week. If you are monitoring the progress of immunization, bleed the rabbits before giving booster injections. -Give booster injections on four consecutive days, giving 0.25, 0.5, 1.0, and 1.5 ml of live antigen that has been washed one time to remove traces of media and adjusted to a concentration of 40 IU/ml. If live antigen is not used, continue to give booster injections with killed antigen, injecting 2.0 ml on each of three consecutive days. -Rest the rabbits for one week and take sufficient blood to produce the trial reagents needed, or exsaguinate the rabbits. Absorption of type a conjugates resulted in the total loss of titer for type a cells. The cross-reactions with type b conjugate were easily eliminated by dilution, with the exception of the cross-reaction with S sanguis JC-43. Bratthalls absorption method eliminated all cross-reactions of the type b conjugate. Absorption of type c conjugate successfully removed the cross-reaction with type e cells; however, the loss of homologous type c titer was so great that this absorption is of limited value. High-titered conjugates for types d and e have been obtained by using batch absorption procedures.

THE DEFINITION AND APPLICATION OF EVALUATION TECHNIQUES AS A GUIDE FOR THE IMPROVEMENT OF FLUORESCENT ANTIBODY REAGENTS

The design of procedures for improving the quality of immunofluorescent conjugates is contingent upon knowledge of the characteristics of ideal reagents. Evaluation techniques are essential for determining how closely existing reagents approach the ideal. Deficiencies can be identified and production methods modified to correct weaknesses. Improvement can be measured by the results of reevaluation of the reagents. Thus, the definition and application of evaluation procedures in a logical sequence is the essence of quality control, which has as its objective the production of ideal reagents. In the past nine months in our laboratory many commercial fluorescent antibody conjugates have been studied by techniques worked out previously but refined and expanded as the study progressed. This report encompasses data obtained from the study of 21 antibacterial conjugates directed against seven species; the reagents were purchased on the open market from seven manufacturers. Several antihuman conjugates have also been examined. Our studies were undertaken t o determine the quality of some widely used commercial conjugates, to identify factors responsible for existing deficiencies, and to suggest methods for improving production. By correlating physicochemical characteristics and biological performance of conjugates, we frequently have been able to detect the cause of performance deficiencies. Thus, physiocochemical data serve as a guide to the probable performance of the reagent. Physicochemical and performance criteria used to describe conjugates are presented in TABLE 1. The performance testing was done independently of the physicochemical testing by different workers in different laboratories. Data were not compared until testing was completed.

Physicochemical characterization of direct fluorescent antibody reagents.

When the data from performance and physicochemical studies of conjugates are combined for analysis, the performance data and specific titers show a direct relationship to the physicochemical data (Table 2). These reagents were prepared from the same lot of antiserum. The specific titers are very misleading without the accompanying data (Table 2). The protein concentrations range from 4 to 10 mg/ml, the F/P ratios from 10 to 30, and CASE shows gamma-globulin to constitute 30 to 100% of the protein. CASE also shows the gamma-globulin F/P ratio to be only 10 to 20. Using these data, we calculated the concentrations of the gamma-globulins and normalized their titers to 10 mg/ml. The value of good fractionation procedures for recovering gamma-globulin and the desirability of obtaining optimal F/P ratios are reflected in the adjusted titers. Physicochemical characterization of conjugates identifies superior and deficient reagents and frequently reveals the cause of inadequate performance. In this way it serves as a quide for improving reagent quality.