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.     

Delayed secondary enrichment for the isolation of salmonellae from broiler chickens and their environment.

Specimens collected from six broiler flocks were cultured for salmonellae by three methods. (i) For direct enrichment, the specimen was homogenized, and 1 ml of the homogenate was inoculated into tetrathionate-brillant green broth; (ii) for preenrichment, liquid specimens and homogenates were incubated at 37 degrees C, and on the next day 1 ml was inoculated into tetrathionate-brillant green broth; and (iii) for delayed secondary enrichment, incubated preenrichment cultures were held at room temperature for 7 to 10 days and then subcultured to fresh tetrathionate-brilliant green broth. All tetrathionate-brilliant green broth cultures were incubated at 42 degrees C for 24 to 48 h before plating. Significantly more isolations of salmonellae were obtained by delayed secondary enrichment than by direct enrichment or preenrichment. Salmonellae were isolated from 417 of 2,283 (18.3%) samples of litter, intestinal contents, and feces cultured by all three methods. Of these positive specimens, direct enrichment detected 208 (49.9%), preenrichment detected 282 (67.6%), and delayed secondary enrichment detected 373 (89.4%). Of 896 specimens of swabs and rinse fluids that were cultured by preenrichment and delayed secondary enrichment, 259 (28.9%) yielded salmonellae. Delayed secondary enrichment detected 254 (98.1%) of these, and preenrichment detected 147 (56.8%). A total of 23 serotypes of salmonellae were identified. The greater effectiveness of delayed secondary enrichment for the isolation of salmonellae was not likely due to the selection of certain serotypes or to an increased inhibition of competing flora.     

Salmonella in surface waters of central New York state.

Six tributary streams and southern Cayuga Lake in central New York state were sampled for the presence of Salmonella on swabs immersed for 4 days. Of a total of 322 swabs, 39% yielded salmonellae. Swabs were cultured in tetrathionate enrichment at 41.5 degrees C. Isolations were made from brilliant green agar. Salmonellae were isolated from many sites on the streams and from some lake sites. Twenty-five serotypes (11 somatic antigen groups) and a distinct biotype of S. typhimurium (H2S negative) were found. Most frequent isolates, in order of decreasing occurrence, were S. typhimurium, S. thompson, S. oranienburg, and S. enteritidis. Several uncommon isolates also appeared. When tested for mouse infectivity, the isolates generally showed little or no virulence. The incidence of clinical salmonellosis among humans was low in the area and the variety of serotypes had not been noted among cattle. The presence of Salmonella in waters ranging in classification from potable to agricultural and industrial indicated the existence of a low level and undefined reservoirs of the bacteria in the region.     

Ambient-temperature primary nonselective enrichment for isolation of Salmonella spp. from an estuarine environment.

A primary, nonselective, ambient-temperature enrichment procedure for isolation of Salmonella spp. is described. The procedure was superior to elevated-temperature selective enrichment for Salmonella when estuarine water samples were examined. Five Chesapeake Bay stations were monitored, over an 8-month period, for the presence of salmonellae. Of 72 water and sediment samples collected, 17 (23.6%) yielded Salmonella spp. Seven serotypes were identified among the isolates. A seasonal pattern was noted for the incidence of the salmonellae. A most probable number procedure, performed by membrane filtration and nonselective enrichment, yielded Salmonella most probable number indices as high as 110 per 100 g of sediment. The results suggest that new methods, such as the one described in this report, are required for isolation of human intestinal pathogens from estuaries and coastal waters.     

Occurrence of Rhodococcus coprophilus and associated actinomycetes in feces, sewage, and freshwater.

Freshwater, sewage, and fecal samples from various sources were examined for Rhodococcus coprophilus, associated actinomycetes, Escherichia coli, and fecal streptococci. Rhodococcus coprophilus was isolated consistently from feces of farm animals, poultry reared in proximity to farm animals, freshwater, and wastewater polluted with animal fecal wastes. It was not isolated from samples of human feces. The ratio of R. coprophilus total actinomycetes was higher in feces from cattle, sheep, ducks, and geese than in specimens from pigs, horses, and fowl. In samples from two freshwater streams polluted by fecal material from farm animals, the ratios of R. copropilus to total actinomycetes were similar to those found in fecal specimens from cattle and sheep. Ratios of fecal coliform to fecal streptococci could not distinguish between fresh human and animal fecal samples and, furthermore, were not reflected in the stream waters polluted by animal fecal material. R. coprophilus has potential in water and dairy bacteriology as a specific indicator organism of fecal pollution due to farm animal wastes.     

Ambient-temperature primary nonselective enrichment for isolation of Salmonella spp. from an estuarine environment.

A primary, nonselective, ambient-temperature enrichment procedure for isolation of Salmonella spp. is described. The procedure was superior to elevated-temperature selective enrichment for Salmonella when estuarine water samples were examined. Five Chesapeake Bay stations were monitored, over an 8-month period, for the presence of salmonellae. Of 72 water and sediment samples collected, 17 (23.6%) yielded Salmonella spp. Seven serotypes were identified among the isolates. A seasonal pattern was noted for the incidence of the salmonellae. A most probable number procedure, performed by membrane filtration and nonselective enrichment, yielded Salmonella most probable number indices as high as 110 per 100 g of sediment. The results suggest that new methods, such as the one described in this report, are required for isolation of human intestinal pathogens from estuaries and coastal waters.     

Rapid Quantitative Method for Salmonella Detection in Polluted Waters

A procedure has been developed for the enumeration of salmonellae in polluted waters using several modifications of existing techniques. Confirmation of salmonellae is achieved within 48 hr. This procedure includes selective enrichment in m-Tetrathionate Broth (22 +/- 1 hr), plating on Brilliant Green Sulfa Agar (20 +/- 1 hr), and confirmation by flagellar (H) agglutination of the growth in a mannosecontaining medium (6 +/- 1 hr). An incubation temperature of 41.5 C was used throughout this procedure. Dilution to extinction techniques (most probable number) were employed to enumerate salmonellae. Large sample volumes were concentrated through the use of membrane filters. This technique proved to be rapid and reliable for the enumeration of salmonellae in water, waste water, and waste-water sludges.     

Examination of Feces from Food Handlers for Salmonellae, Shigellae, Enteropathogenic Escherichia coli, and Clostridium perfringens

Duplicate fecal specimens from food handlers were collected in Louisiana. One set of specimens was examined immediately for salmonellae and shigellae by the Central Laboratory of the Louisiana State Board of Health in New Orleans; the other set was shipped to the Food Microbiology Unit at the Robert A. Taft Sanitary Engineering Center in Cincinnati, Ohio, where it was examined for enteropathogenic Escherichia coli (EEC) and Clostridium perfringens. A total of 219 specimens were examined by both laboratories. None yielded salmonellae or shigellae; 171 (78.1%) yielded C. perfringens; 175 (79.9%) yielded E. coli; and 14 (6.4%) yielded EEC. The 14 isolates of EEC were distributed among eight serotypes; one specimen yielded two serotypes. Multiple isolations of C. perfringens strains (two to four) were made from 64 (37.4%) of the specimens, and a total of 244 strains were isolated and studied for identifying characteristics. Of the total, only 87 (35.5%) could be identified serologically by a battery of 67 antisera; only 4 (1.6%) possessed the characteristics of the English “food-poisoning type.” The hemolytic activity on agar containing horse, ox, or sheep blood showed that 140 (57.1%) were “hemolytic,” 81 (33.1%) were “nonhemolytic,” and 23 (9.8%) gave varied results. Only 12 (4.9%) of the strains produced spores that resisted boiling for 30 min or more.     

A comparison of isolation procedures for salmonellas from polluted water using two forms of Rappaport's medium

F ricker , C.R. 1984. A comparison of isolation procedures for salmonellas from polluted water using two forms of Rappaport's medium. Journal of Applied Bacteriology 56 , 305–309.
The efficiency of Rappaport's broth (RB10) and Rappaport's broth containing novobiocin (NRB10) were compared for the isolation of salmonellas from polluted water, both as direct enrichment media and after pre-enrichment in buffered peptone water. Ninety samples were examined and 41 were found to contain salmonellas by at least one of the procedures used. Direct inoculation of the sample into RB10 resulted in the recovery of salmonellas from only 29.3% of the samples found to be positive. The use of NRB10 as a direct enrichment medium increased the percentage recovery to 78.0% of the total positive samples. Pre-enrichment in buffered peptone water allowed the recovery of salmonellas from a total of 41 samples whereas direct enrichment recovered them from only 32. No significant difference was demonstrated in the efficiencies of RB10 and NRB10 in recovering salmonellas after pre-enrichment in buffered peptone water. Three selective agars were used; no significant difference in their ability to recover salmonellae was demonstrated.     

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.     

Aquarium pets as a source of antibiotic-resistant salmonellae.

Thirteen serotypes of Salmonella isolated from imported ornamental aquarium frogs, snails, and their waters were shown to be multi-drug-resistant. Among the resistances exhibited were resistance to gentamicin, sulfamethoxazole-trimethoprim, cephalothin, and nalidixic acid. Frog isolates displayed eight different patterns and snails isolates had two different resistance patterns. The most common serotype, Salmonella typhimurium, was resistant to 18 antibacterials while other salmonellae were resistant to 9 to 16 antibacterials. Resistances in S. typhimurium and S. bovis-morbificans were conjugative and a number of R plasmids participated in the resistance. The plasmid-mediated resistance in S. typhimurium was stable and the levels of resistance conferred were markedly higher than in the other salmonellae tested. Resistance of other serotypes was non-conjugative and resistance to the beta-lactam antibiotics was unstable.     

Diversity of rotifers from northeastern U.S.A. bogs with new species records for North America and New England

In order to assess the effects of pollution on recruitment of brown trout (Salmo trutta f. fario L.) exposure experiments with brown trout early life stages were performed in two differently contaminated small streams near Stuttgart, southwest Germany, and in a control situation in the laboratory. Pollution in the more polluted stream is mainly due to effluents of municipal sewage treatment plants and to the influx of surface waters from areas of intense agriculture activity. Additionally, the water carries high particle loads, especially after rainfall events. Water quality of the less polluted stream is occasionally influenced by agriculture activity in the vicinity. For the exposure of trout early life stages incubators were used, which allowed frequent examinations without serious disturbance of the exposed organisms and thus the development could be monitored over time. Compared to the less polluted stream and the control, in the more polluted stream high mortality rates, the lowest hatching success, and lowest values for growth occurred. In the less polluted stream, mortality rates were comparably low in prehatching stages, but high in hatchlings and juveniles due to heavy infestations with protozoic ectoparasites Chilodonella cyprini (Moroff, 1902) and Ichthyobodo necator (Henneguy, 1883). However, pollution-related effects were not detected in this stream. Differences in developmental rates between the different treatments were correlated with different water temperatures in the two streams and the control. For the more polluted stream, previous studies have indicated embryotoxic potentials mediated by pollutants. The present study indicates that in this steam, additionally, fine solids which infiltrate stream gravels may seriously affect development of brown trout early life stages.     

Salmonella Contamination in a Poultry-Processing Plant

Bacteriological examination of 1,427 samples from a poultry-processing plant over a 2-year period yielded 202 (14.2%) cultures positive for salmonellae. The results indicate that contamination is reduced by washing procedures within the plant but that recontamination of the carcasses occurred in at least two different stages of processing, i.e., during evisceration and chilling. There was evidence of spread of salmonellae from flock to flock during the serial processing of flocks, but the spread was usually not extensive. The serotypes of salmonellae isolated in this study were similar to those of chicken origin reported from other areas of the country.     

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.     

Microbiological Quality of Protein Feed Supplements Produced by Rendering Plants

Samples of protein feed supplements produced by rendering plants were examined for salmonellae, total aerobic bacterial counts, coliform counts, and enterococci. Isolations of salmonellae were more frequent from products with high counts; however, 6% of the samples with total counts of less than 1,000 per gram and 14% of the samples with coliform counts of less than 1 per gram contained salmonellae. Serotypes of Escherichia coli which have been associated with disease in domestic animals and poultry were also isolated from products. Although the distribution of serotypes of salmonellae isolated from environmental swabs and flies was similar to that isolated from products, the isolation of several serotypes from flies which had not been isolated in plants suggested that flies may be a potential source of contamination.     

Occurrence and distribution of culturable enteroviruses in wastewater and surface waters of north‐eastern Spain

Aims: Update information regarding occurrence and levels of culturable enteroviruses in several types of surface polluted waters in north‐eastern Spain and determine the proportion of the different species and serotypes. Methods and Results: The best procedures on hand in our laboratory for concentrating and quantifying culturable enteroviruses from different water sample types were used. Sequencing was used for typing the virus isolates. Geometric means of enteroviruses densities expressed in plaque forming units per litre were 968 in raw sewage, 12·51 in secondary effluents, 0·017 in tertiary effluents, 0·4 in river water and 0·36 in seawater. Enterovirus densities in wastewater revealed certain seasonality with a maximum at the end of spring – beginning of the summer. Coxsackievirus B, and amid them serotype CB4, were the most abundant species and serotypes detected. Conclusions: Densities of enteroviruses in different north‐eastern Spain surface waters are similar to those present in industrialized countries with temperate climate. No wild polioviruses were detected. Distribution of species showed a clear prevalence of coxsackieviruses. Significance and Impact of the Study: Information regarding enteroviruses in this geographical area provides valuable information to estimate the risk of enteroviruses transmission through water and for complementing clinical epidemiological data.     

Salmonellae Associated with Further-processed Turkey Products

"Further-processed" turkey products, prepared from chilled, eviscerated, and thawed carcasses at two commercial turkey-processing plants, were evaluated, for the presence of salmonellae. These organisms were isolated from swab samples from 12% of chilled, eviscerated turkey carcasses, 27% of finished products, and 24% of processing equipment. The same serotypes as those found throughout a plant on any one visit were recovered from 31% of rinse-samples taken from hands and gloves of processing personnel. Salmonellae were found in samples taken on 37 of 48 visits; a greater number of recoveries were made on days when freshly killed turkeys were processed (87%) than when frozen-defrosted carcasses were processed (59%). The predominant serotype isolated from meat and environment usually changed from visit to visit. Salmonella sandiego and Salmonella anatum were the most frequent among 23 serotypes recovered. Most of the isolated serotypes are commonly associated with turkeys and have been incriminated as causative agents of human salmonellosis. The implication is that further-processed turkey products, if inadequately cooked by the consumer and if improperly refrigerated between the time of manufacture and consumption, could directly transmit salmonellae. These same products might also contaminate other foods by introducing salmonellae into food-preparation areas.     

Isolation of Salmonellae from Sewage with a New Procedure of Enrichment

Forty samples of sewage on Moore's swabs were examined for the presence of salmonellae. They were first pre-enriched in buffered peptone water. From each pre-enrichment, three enrichments were made: (1) in a new, considerably modified, formula of Rappaport medium (R 10) incubated at 43 °C (R 10/43 °C), (2) in the usual formula (R25) of the same medium at 37 °C (R25/37 °C) and (3) in Muller-Kauffmann's tetrathionate broth at 43 °C (MK/43 °C). Practically the same numbers of swabs were found positive by the first two enrichment procedures, 38 and 39 respectively, while only 17 were found positive by the MK procedure. The R10/43 °C method was superior to the two other procedures; it yielded 103 strains of salmonellae as against 82 with the second Rappaport procedure, and only 25 with the MK/43 °C technique. A similar observation was made concerning the frequency of isolation of different serotypes by the three procedures; the number of the isolated serotypes was 24, 19 and 11, respectively. The new R 10/43 °C method of enrichment had also a much stronger inhibitory effect on the competing bacteria than the two other procedures of enrichment used.     

Incidence of Salmonellae in Dressed Broiler-Fryer Chickens

Salmonellae were isolated from 72 of 264 broiler-fryer type chickens that had been purchased in retail stores in the Lafayette, Ind., area in 1963. Meat from the tail area and giblet portions were used in sampling. Equal numbers of dressed whole and cut-up birds were positive for salmonellae. Thirteen different serotypes were identified, the more common being Salmonella infantis, S. reading, and S. blockley. Incubation at 43 C of the blended sample in Selenite-F Enrichment broth containing cystine gave a larger number of recoveries than did incubation at 37 C. There was no significant difference between the means for the birds that yielded salmonellae and those that did not in the locally processed group, when compared for numbers of aerobic microorganisms at 37 C, coliforms, or most probable number of enterococci. In a comparison of poultry processed in-state by the five processors included in the study with that processed out-of-state, there was a general trend for a larger number of positive specimens in the locally produced group. The fall season was an exception.