Probeliatrade mark PCR system for rapid detection of Salmonella in milk powder and ricotta cheese

The Probeliatrade mark Salmonella sp. PCR amplification and detection kits (Sanofi Diagnostics Pasteur, Marnes La Coquette, France) were evaluated for the rapid and specific detection of Salmonella agona artificially inoculated into skim milk powder and ricotta cheese. The Probeliatrade mark results were compared with those obtained using the Australian Standard Method. Using a pure culture of Salm. agona, the detection limit of Probeliatrade mark was between 8 and 79 cfu ml-1, equivalent to 0.2-2 cfu per PCR reaction. Detection of Salm. agona inoculated in skim milk powder (at 5-10 cfu g-1, stored at 5, 15 or 25 degrees C) and ricotta cheese (at 1-2, 10-20 and 100-200 cfu per 25 g) was effected by using non-selective enrichment prior to the PCR determinations. For all of the 40 milk powder samples and 12 ricotta cheese samples, the Probeliatrade mark results were consistent with those using the Australian Standard Method. Using Probeliatrade mark, Salmonella was detected to genus level in the dairy products within 24-28 h, whereas the cultural technique required 3-4 d for presumptive positive isolates and further time for confirmation.     

The detection of Salmonella using a combined immunomagnetic separation and ELISA end-detection procedure

The aim of this study was to develop a rapid immunoassay to detect Salmonella bacteria. Skimmed milk powder (SMP) in buffered peptone water was inoculated with six Salmonella strains (Salm. typhimurium, Salm. virchow, Salm. enteritidis, Salm. give, Salm. ealing and Salm. arizonae) at three inoculum levels (about 2-200 cfu 25 g(-1) SMP) and incubated (37 degrees C) overnight. Heat-treated salmonella cells were immobilized on paramagnetic particles and detected within 3 h using the Salmonella genus-specific monoclonal antibody M105 in a microtitre plate based assay. The rapid Salmonella detection method combining immunomagnetic separation and ELISA had a total isolation and detection time of less than 24 h, which is significantly shorter than the conventional techniques requiring 72-96 h. The technique had a sensitivity limit of 10(5)-10(6) cfu ml(-1).     

Survival and heat resistance of Salmonella enterica and Escherichia coli O157:H7 in peanut butter

Significant differences (P < 0.05) were found between the survival rates of Salmonella enterica and Escherichia coli O157:H7 in peanut butter with different formulations and water activity. High carbohydrate content in peanut butter and low incubation temperature resulted in higher levels of bacterial survival during storage but lower levels of bacterial resistance to heat treatment.     

Survival of faecal indicator bacteria in bovine manure incorporated into soil

AIMS: Survival of Escherichia coli and enterococci was evaluated in bovine manure incorporated into two Wisconsin soils. METHODS AND RESULTS: Silty clay loam (SCL) and loamy sand (LS) were mixed with fresh bovine manure, exposed daily to 10 h at 22 degrees C/14 h at 9 degrees C, and watered weekly for 12 weeks. Escherichia coli numbers increased 1-2 log cfu g(-1), then decreased < 1 and about 2 log cfu g(-1) in SCL and LS, respectively. Enterococci numbers rose less and then declined faster than those of E. coli. Watering intervals of 3, 7 and 14 days were evaluated in weeks 13-19, but did not affect the slow decline in numbers of E. coli or enterococci. CONCLUSION: Escherichia coli and enterococci may survive at least 19 weeks at 9-21 degrees C in bovine manure/soil, with E. coli surviving better. SIGNIFICANCE AND IMPACT OF THE STUDY: Quantification of E. coli or enterococci in late spring/early summer soil may be useful in indicating recent application of bovine manure.     

Growth of Escherichia coli O157:H7 during sprouting of alfalfa seeds

AIMS: Escherichia coli O157:H7 was monitored daily during sprouting of alfalfa seeds inoculated at high (3.92 log10 cfu g(-1)) and low (1.86 log10 cfu g(-1)) levels to assess the extent of pathogen growth during production. METHODS AND RESULTS: Sprouts and rinse water were tested by direct and membrane filter plating on modified sorbitol MacConkey agar and BCM O157:H7(+) agar; the antibody-direct epifluorescent filter technique; and rapid immunoassays. The pathogen reached maximum populations after one and two days of sprouting seeds inoculated at high and low levels, respectively; in either case, populations of 5-6 log10 cfu g(-1) were reached. Detection limits of two rapid immunoassays, Reveal and VIP, without enrichment were determined to be 5-7 log10 cfu ml(-1). CONCLUSION: These results show the ability of E. coli O157:H7 to grow to high levels during sprouting; however, because these levels may be below detection limits, it is necessary to include enrichment when monitoring sprout production for E. coli O157:H7 by the rapid test kits. SIGNIFICANCE AND IMPACT OF THE STUDY: The data indicate that sprouts may harbor high levels of pathogens. The appropriate use of rapid test methods for pathogen monitoring during sprouting is indicated.     

Determination of aflatoxin levels in peanut butter using HPLC and ELISA procedures: Inter-laboratory comparison

Mycotoxin Research - Six laboratories analyzed portions of the same aqueous acetonitrile extracts of three peanut butters for aflatoxin concentrations by an HPLC procedure (using immunoaffinity...     

Aflatoxins in peanut butter in Khartoum State,Sudan

Forty-three peanut butter samples from Khartoum State, Sudan, were analyzed for aflatoxins (AFs, AFB₁ + AFB₂ + AFG₁ + AFG₂) using high performance liquid chromatography (HPLC) with fluorescence detection after extraction with methanol:water (8:1, v/v) and clean-up using chloroform. All samples were contaminated with AFs, with total AF levels ranging between 26.7 and 853 μg/kg, and a mean total AF level of 287 ± 200.5 μg/kg. The highest concentrations were found for AFB₁, (28 positive samples, maximum 534 μg/kg), while AFG₁ was most frequently detected (43 positive samples, maximum 401 μg/kg). AFB₂ (42 positive samples, maximum 3.2 μg/kg) and AFG₂ (4 positive samples, maximum 30 μg/kg) were also present in these samples. The mean AF contamination levels found in this study exceeded by far all international regulations concerning maximum levels for this group of toxins. From the data, it is concluded that the levels of AF contamination in peanut butter from the Kartoum area are quite alarming, and may pose serious health hazards to consumers. Therefore, an intervention strategy to manage AF in peanut butter is urgently needed.     

Nisin and ALTATM 2341 inhibit the growth of Listeria monocytogenes on smoked salmon packaged under vacuum or 100% CO2

The effects of nisin and ALTA 2341 on the growth of Listeria monocytogenes were assessed on smoked salmon packaged under vacuum or 100% CO2. Smoked salmon slices (pH 6.3) were inoculated with a cocktail of seven L. monocytogenes isolates at a level of approximately 2.5 log10 colony forming units (cfu) g-1. After inoculation, the surface of the smoked salmon slices was treated with either nisin (400 or 1250 IU g-1) or ALTA 2341 (0.1 or 1%). The smoked salmon was packaged and stored at 4 degrees C (28 d) or 10 degrees C (9 d). On untreated vacuum-packaged smoked salmon, L. monocytogenes grew by 3.8 log10 cfu g-1 at 4 degrees C and 5.1 log10 cfu g-1 at 10 degrees C. Growth was reduced on nisin- and ALTA 2341-treated vacuum-packaged smoked salmon. On the nisin-treated samples, L. monocytogenes increased by 2.5 (400 IU g-1) and 1.5 (1250 IU g-1) log10 cfu g-1 at 4 degrees C, and by 4.3 (400 IU g-1) and 2.7 (1250 IU g-1) log10 cfu g-1 at 10 degrees C. With the ALTA 2341-treated samples, L. monocytogenes increased by 2.8 (0.1%) or 1.6 (1.0%) log10 cfu g-1 at 4 degrees C, and 3.3 (0.1%) or 3.6 (1.0%) log10 cfu g-1 at 10 degrees C. The growth of L. monocytogenes was retarded by packaging the smoked salmon in 100% CO2. On untreated smoked salmon, only a 0.8 log10 cycle increase was observed at 10 degrees C. Under all the other conditions tested with 100% CO2, L. monocytogenes was detected but growth was prevented.     

Control of foodborne pathogens during sufu fermentation and aging

Control of the foodborne pathogens Escherichia coli O157:H7, Salmonella typhimurium, Staphylococcus aureus, and Listeria monocytogenes during sufu fermentation was evaluated. Before fermentation, pathogens were inoculated onto tofu (substrate for sufu) at 5 log cfu/g or 3 log cfu/g, and starter culture (Actinomucor elegans) was inoculated at 3 log cfu/g. After 2 days of fermentation at 30 degrees C, the four pathogens reached 7 to 9 log cfu/g, and the mold count reached 6 to 7 log cfu/g. After fermentation, sufu samples were aged in a solution of 10% alcohol + 12% NaCl. After 1 month of aging, the total bacterial count was 6 to 7 log cfu/g, but all foodborne pathogens and mold were reduced to nondetectable levels. The total bacterial count decreased after aging for 2 months and 3 months, but the differences were not significant (P > 0.05) compared with the count after 1 month. Microorganism in experimental sufu from different aging periods and in commercial sufu were compared. A total of 270 isolates were purified and identified by the BBL Crystal Identification System. From the experimental sufu samples, 49 Bacillus spp. (20.4%), 167 Enterococcus spp. (69.6%), 6 Shewanella putrefaciens (2.4%), and 18 miscellaneous gram-negative bacilli (7.5%) were identified. From commercial sufu samples, 17 Bacillus spp. (56.7%), 2 Enterococcus durans (6.7%), 5 miscellaneous gram-negative bacilli (16.7%), 5 Corynbacterium aquaticum (16.7%), and 1 Shewanella putrefaciens (3.3%) were obtained. Although the longer aging period did not significantly decrease the total bacterial count, it may help in the development of sufu flavor. This study showed that sufu fermentation and aging can control common foodborne pathogens, so sufu is a safe product even though its preparation does not include pasteurization.     

Survival of Escherichia coli O157:H7 in farm water: its role as a vector in the transmission of the organism within herds

AIMS: The study aimed to investigate the survival characteristics of Escherichia coli O157:H7 in farm water (FW), and in sterile distilled municipal water (SDW), stored outdoors under field conditions, with or without the addition of faeces (1% w/v), in a farmyard shed and the laboratory at 15 degrees C. METHODS AND RESULTS: Water samples were inoculated with E. coli O157:H7 at 10(3) and 10(6) ml(-1), and sampled over a 31-day period. In FW stored outdoors in a field, E. coli O157:H7 survived for 14 days at temperatures <15 degrees C, at both inoculation levels, while in the laboratory at 15 degrees C, the organism was still detectable at low levels (<1 log10 cfu ml(-1)) after 31 days. The addition of bovine faeces to water outdoors (1% w/v) resulted in survival for 24 days. In SDW inoculated at 10(6) ml(-1) and stored in the laboratory (15 degrees C), only a 2.5 log reduction was observed after 31 days, while the organism could not be detected after 17 days in the field. Preliminary screening of water samples stored outdoors isolated a bacterium which exhibited antimicrobial activity towards E. coli O157:H7. CONCLUSIONS: The survival of E. coli O157:H7 observed in this study illustrates the potential of farm water to act as a vehicle in the transfer of the organism across a herd. SIGNIFICANCE AND IMPACT OF THE STUDY: The difficulty in extrapolating results from controlled laboratory situations to on-farm conditions is also highlighted in this study.     

Survey of aflatoxin-producing fungi in certain fermented foods and beverages in Thailand

Aflatoxin-producing fungi were found in fermented foods and beverages: fermented rice (kaomak), soybean sauce (taotjo), peanut butter, soy sauce (shoyu), Thai red and white wine, and rice sugar wine. These foods were extracted directly and tested for aflatoxins by thin-layer chromatography (TLC) and high pressure liquid chromatography (HPLC).Four strains of aflatoxin-producing fungi were isolated from peanut butter, taotjo, and shoyu. Direct extracts of 10% of the peanut butters tested and 5% of the kaomak tested contained large amounts of aflatoxins. The HPLC procedure used in this experiment utilized chloroform-ethyl acetate (31)     

Salmonella enteritidis and aerobic mesophiles in inoculated poultry sausages manufactured with high-pressure processing

Salmonella enteritidis-inoculated poultry sausages were pressurized at 500 MPa by combining different times (10 and 30 min) and temperatures (50, 60 and 70 degrees C) or heat treated with the same temperature-time combinations and a standard cooking (75 degrees C for 30 min). Counts of Salm. enteritidis and mesophilic bacteria were determined. Most pressure treatments generated statistically higher reductions than the corresponding heat treatments alone. Lethalities of about 7.5 and 6.5 log cfu g(-1) for Salm. enteritidis and mesophiles, respectively, were found in pressurized sausages. There was no significant difference in counts between pressurization at 60 degrees C for 30 min or at 70 degrees C and the standard cooking. High-pressure processing is a suitable alternative method in poultry sausage manufacture.     

Development of a surface adhesion immunofluorescent technique for the rapid detection of Salmonella spp. from meat and poultry

A rapid method based on bacterial adhesion was developed for the detection of Salmonella in an enriched meat system. Minced beef samples inoculated with Salm. enteritidis (10 cfu g-1) were incubated overnight (18 h) at 37 degrees C in buffered peptone water. Salmonella enteritidis cells were isolated from the enriched meat sample by surface adhesion onto a polycarbonate membrane attached to a glass slide. The organisms attached to this polycarbonate membrane were subsequently visualized using immunofluorescent microscopy. The technique had a detection level of log10 3.5 Salmonella ml-1. The surface adhesion immunofluorescent technique correlated well with Salmonella plate counts (r2 = 0.99). Application of the rapid method to retail beef and poultry samples (n = 100) confirmed the correlation between this technique and traditional microbiological procedures. Thirty-one retail samples were reported positive for Salmonella species. No false positives or negatives were recorded for the rapid method.     

Effect of packaging conditions on the growth of micro-organisms and the quality characteristics of fresh mushrooms (Agaricus bisporus) stored at inadequate temperatures

Mushrooms were packed in two polymeric films (perforated and non-perforated PVC) and stored at 17 degrees C and 25 degrees C. The carbon dioxide and oxygen content inside the packages, aerobic mesophiles, Pseudomonas spp., faecal coliforms, Escherichia coli, anaerobic spores and major sensory factors (colour, texture, development stage and presence of moulds) were determined. The non-perforated packages had the highest contents of CO2 (6-7%), the lowest contents of O2 (0.013-0.17%) and the most desirable quality parameters (texture, development stage and absence of moulds). Pseudomonas spp. counts were around 1 logarithmic unit lower in mushrooms packaged in non-perforated film as the O2 concentrations were lower than in perforated film. The mushrooms themselves were inoculated with an enterotoxin A-producing strain of Staphylococcus aureus, packaged in overwrapped trays and stored at 17 and 25 degrees C. Staphylococcus aureus did not grow in the samples stored at 17 degrees C. Only slight growth was observed in mushrooms packaged with non-perforated film after 1 day at 25 degrees C. No enterotoxin was detected in any package. Faecal coliform counts were <2 log cfu g(-1). Escherichia coli was not isolated in any of the samples. At 25 degrees C, counts of anaerobic spores of around 2 log cfu g(-1) were detected in those mushrooms packaged in non-perforated film.     

Efficacy of chlorine and heat treatment in killing Salmonella stanley inoculated onto alfalfa seeds and growth and survival of the pathogen during sprouting and storage.

The efficacy of chlorine and hot water treatments in killing Salmonella stanley inoculated onto alfalfa seeds was determined. Treatment of seeds containing 10(2) to 10(3) CFU/g in 100-micrograms/ml active chlorine solution for 5 or 10 min caused a significant (P < or = 0.05) reduction in population, and treatment in 290-micrograms/ml chlorine solution resulted in a significant reduction compared with treatment in 100 micrograms of chlorine per ml. However, concentrations of chlorine of up to 1,010 micrograms/ml failed to result in further significant reductions. Treatment of seeds containing 10(1) to 10(2) CFU of S. stanley per g for 5 min in a solution containing 2,040 micrograms of chlorine per ml reduced the population to undetectable levels (< 1 CFU/g). Treatment of seeds in water for 5 or 10 min at 54 degrees C caused a significant reduction in the S. stanley population, and treatment at > or = 57 degrees C reduced populations to < or = 1 CFU/g. However, treatment at > or = 54 degrees C for 10 min caused a substantial reduction in viability of the seeds. Treatment at 57 or 60 degrees C for 5 min appears to be effective in killing S. stanley without substantially decreasing germinability of seeds. Storage of seeds for 8 to 9 weeks at 8 and 21 degrees C resulted in reductions in populations of S. stanley of about 1 log10 and 2 log10 CFU/g, respectively. The behavior of S. stanley on seeds during soaking germination, sprouting, and refrigerated storage of sprouts was determined. An initial population of 3.29 log10 CFU/g increased slightly during 6 h of soaking, by about 10(3) CFU/g during a 24-h germination period, and by an additional 10 CFU/g during a 72-h sprouting stage. A population of 10(7) CFU/g of mature alfalfa sprouts was detected throughout a subsequent 10-day storage period at 5 degrees C. These studies indicate that while populations of S. stanley can be greatly reduced, elimination of this organism from alfalfa seeds may not be reliably achieved with traditional disinfection procedures. If S. stanley is present on seeds at the initiation of the sprout production process, populations exceeding 10(7) CFU/g can develop and survive on mature sprouts exposed to handling practices used in commercial production and marketing.     

Development of a surface adhesion immunofluorescent technique for the rapid isolation of Listeria monocytogenes and Listeria innocua from meat

The use of a novel surface adhesion technique to isolate Listeria monocytogenes and Listeria innocua from an enrichment meat system was developed. Minced beef samples inoculated with L. monocytogenes (10 cfu g(-1)) were incubated at 30 degrees C for 14-18 h in a suitable enrichment broth. Listeria monocytogenes cells were isolated from the enriched meat sample by surface adhesion onto a polycarbonate membrane which was attached to a glass microscope slide. The Listeria cells on the membrane were subsequently visualized using an immunofluorescent microscopy procedure. The antibody used in this technique reacts with L. monocytogenes and L. innocua. The technique was demonstrated to have a detection level of log10 3.11 cfu ml(-1). There was excellent correlation (r2 = 0.98) between the counts obtained by this surface adhesion immunofluorescent (SAIF) technique and counts obtained using traditional methods, i.e. plate counts on PALCAM. When the regression equation relating the rapid and standard methods was validated using the data from 50 retail beef mince samples, an rsd value of +/- 0.25 was obtained. No false-negative or false-positive results were recorded for L. monocytogenes or L. innocua species using the SAIF technique.     

Production of butter fat rich in trans10-C18:1 for use in biomedical studies in rodents

Trans fatty acids are suspected to be detrimental to health, particularly to cardiovascular function. Trans fatty acids include a wide range of fatty acids, with isomers of C18:1, conjugated and non-conjugated C18:2 as major components. A vaccenic acid (trans11-C18:1) + rumenic acid (cis9,trans11-CLA)-rich butter has been shown previously to exhibit health beneficial effects, but less is known concerning another trans-C18:1 present in hydrogenated vegetable oil-based products and sometimes in milk fat, the trans10-isomer. The present experiment was conducted to produce butters from milk of variable fatty acid composition for use in biomedical studies with rodents, with the overall aim of evaluating the specific effect of trans10-C18:1 and trans11-C18:1 + cis9,trans11-CLA on cardiovascular function. Milks from lactating dairy cows fed two types of maize-based diets supplemented (5% of dry matter)--or not--with sunflower oil were collected, and used to manufacture butters either rich in trans10-C18:1 (14% of total fatty acids, 64.5% of fat content) or rich in trans11-C18:1 + cis9,trans11-CLA (7.4 and 3.1% of total fatty acids, respectively, 68.5% of fat content), or with standard fatty acid composition (70% of fat content). Additionally, total saturated fatty acid percentage was reduced by more than one third in the enriched butters compared with the standard butter. An understanding of the role of nutrition on milk fatty acid composition in cows allows for the production of dairy products of variable lipid content and composition for use in biomedical studies in animal models and human subjects.     

Survival of Campylobacter jejuni on beef trimmings during freezing and frozen storage

AIMS: To investigate the survival of two animal isolates of Campylobacter jejuni on beef trimmings during freezing and frozen storage. METHODS AND RESULTS: Meat packs inoculated with 10(3) or 10(6) cfu g(-1) of either strain of C. jejuni were frozen to -18 degrees C, and sampled at regular intervals over 112 d storage to determine Campylobacter numbers and sublethal injury. For both strains and inoculation levels the numbers of Campylobacter decreased in the first 7 d of storage by ca. 0.6-2.2 log cfu g(-1) and then remaining constant over the remainder of the storage trial, with neither isolate exhibiting sublethal injury. CONCLUSIONS: Despite an initially significant decrease in number, these pathogens were able to survive standard freezing conditions in meat, but did not exhibit sublethal injury. SIGNIFICANCE AND IMPACT OF THE STUDY: Strict hygiene and/or the implementation of decontamination technologies are recommended as a means to assure the safety of meat with respect to this pathogen.     

Survival of Escherichia coli O157:H7 and Salmonella typhimurium inoculated on chicken by aqueous chlorine dioxide treatment

Inactivation of Escherichia coli O157:H7 and Salmonella typhimurium was evaluated on inoculated chicken by aqueous chlorine dioxide (ClO2) treatment. Chicken samples were inoculated with 6-7 log CFU/g of Escherichia coli O157:H7 and Salmonella typhimurium, respectively. The chicken samples were then treated with 0, 50, and 100 ppm of ClO2 solution and stored at 4 +/- 1 degrees C. Aqueous ClO2 treatment decreased the populations of the pathogenic bacteria on the chicken breast and drumstick. In particular, 100 ppm ClO2 treatment on the chicken breast and drumstick reduced Escherichia coli O157:H7 and Salmonella typhimurium by 1.00-1.27 and 1.37-1.44 log CFU/g, respectively. Aqueous ClO2 treatment on the growth of the bacteria was continuously in effect during storage, resulting in the decrease of the populations of Escherichia coli O157:H7 and Salmonella typhimurium. These results suggest that aqueous ClO2 treatment should be useful in improving the microbial safety of chicken during storage.     

The survival characteristics of a non-toxigenic strain of Escherichia coli O157:H7

The survival characteristics of a non-toxigenic, antibiotic-resistant strain of Escherichia coli O157:H7 in bovine faeces were investigated. Faecal samples were inoculated with 10(8-9) cfu g-1 of the organism and (i) stored in closed plastic containers at 10 degrees C, (ii) stored in closed plastic containers placed outside or (iii) decanted onto the surface of grazing land. Recovery and enumeration on Sorbitol MacConkey Agar (SMAC) and Tryptic Soya Agar (TSA) revealed that the E. coli O157:H7 numbers in both enclosed samples (i and ii) had decreased by 4.5-5.5 log10 cfu g-1 within 99 d. Numbers in samples decanted onto grassland (iii) decreased by 4.0-5.0 log10 cfu g-1 within 50 d but the organism was still detectable in the surrounding soil for up to 99 d. Persistence of E. coli O157:H7 in bovine faeces and contaminated pastures may therefore be an important factor in the initial infection and re-infection of cattle.