A procedure for extracting staphylococcal enterotoxin from foods at elevated temperatures and low centrifugal forces

Various low relative centrifugal forces (RCFs) were tested for their effectiveness in ‘cleaning up’ extracts of staphylococcal enterotoxin from food homogenates. RCFs of at least 10 000 × g were effective if centrifugations were for 30 min periods or longer. Below 8000 × g, and down to about 1000 × g, the cleaning up of extracts was effective only if centrifugation was preceded by filtration of food homogenates. Investigations of the thermal stability of enterotoxin during cleaning up of extract at higher than refrigeration temperatures showed that centrifugation temperatures of up to 40°C had little adverse effect on the serological activity of the toxin. Based on these findings a procedure was devised for extracting enterotoxin from foods using basic bench top centrifuge at ambient temperatures. Separation of enterotoxin from food proteins in the extract was achieved by carboxymethyl cellulose-column chromatography. Levels of enterotoxin A detectable by the microslide gel double diffusion method following extraction by this procedure were 2.5 ng/ml for milk, 2.5 ng/g for yogurt and 5 ng/g for cheese and corned beef.     

Sensitive detection of multiplex toxins using antibody microarray

Using a newly developed fluorescent nanoparticle (NP) that gives rise to a high-intensity and stable fluorescent light, a sensitive antibody (Ab) microarray assay system has been developed for specific detection of bioterrorism agents, as exemplified by ricin, cholera toxin (CT), and staphylococcal enterotoxin B (SEB). The Ab microarray uses a sandwich format that consists of capture Abs, analytes (toxins), biotinylated detection Abs, and avidin-conjugated NP. In all three cases, polyclonal Abs (pAbs) displayed superiority over monoclonal antibodies (mAbs) in capturing toxins on microarray slides even when the pAbs and mAbs had similar affinity as determined by enzyme-linked immunosorbent assay (ELISA). The detection system was successfully used to detect toxins spiked in milk, apple cider, and blood samples. We were able to detect ricin at 100 pg/ml in buffer and at 1 ng/ml in spiked apple cider or milk, whereas CT and SEB were detected at 10 pg/ml in buffer and 100 pg/ml in spiked apple cider or milk. High specificities were also demonstrated in the detection of mixed toxin samples with similar sensitivities. The matrix effect of blood samples on the detection of mixed toxins seems to be minimal when the toxin concentration is at or above 100 ng/ml. The current study highlights the significant role of pAb and NP in increasing selectivity and sensitivity of toxin detection in a microarray format.     

Protein A insensitive ELISA detection of staphylococcal enterotoxin B

A sandwich enzyme-linked immunosorbent assay to detect staphylococcal enterotoxin B was developed using rat monoclonal antibodies as capture antibodies and as a biotinylated conjugate. This test was sensitive, less than 1 ng/ml of enterotoxin B was detected and interference by protein A was prevented by the use of rat monoclonal antibodies of the IgG2a isotype which were insensitive to protein A even at concentrations greater than 1000 ng/ml.     

Production of Enterotoxin A in Milk

Enterotoxin A production in milk was studied by use of variables of milk quality, initial numbers of enterotoxigenic staphylococci, incubation temperature, and time. In both raw and pasteurized milks having a low total viable count, enterotoxin was detected in minimal incubation times of 6 to 9 hr at 35 C, 9 to 12 hr at 30 C, 18 hr at 25 C, and 36 hr at 20 C, after inoculation with 10(6)Staphylococcus aureus cells per ml. When similar milks were inoculated with 10(4)S. aureus cells per ml, enterotoxin was detected in 12 hr at 35 C, 18 hr at 30 C, 24 to 36 hr at 25 C, and 48 to 96 hr at 20 C. In high-count raw milk, enterotoxin was detected only in samples inoculated with 10(6)S. aureus cells per ml and incubated at 35 C. Generally, a concentration of 5 x 10(7)S. aureus cells per ml of milk was reached before enterotoxin A was detected.     

Occurrence of aflatoxin M<Subscript>1</Subscript> in commercial pasteurized milk samples in Sari,Mazandaran province,Iran

The frequency and levels of aflatoxin M₁ (AFM₁) in pasteurized milk samples in Sari, located in Mazandaran province, Iran, were determined by enzyme immunoassay. Seventy-six samples of pasteurized milk from different retail stores were randomly collected over four seasons during the year 2015. AFM₁ contamination was detected in all milk samples. The mean concentration of aflatoxin M₁ was 65.8 ng/l, with a range of 11.7–106.6 ng/l. The highest AFM₁ level was detected in milk samples collected during spring. Forty-six (60.53 %) samples had AFM₁ levels that exceeded the maximum acceptable levels (50 ng/l) recommended by the European Union (EU). Comparison of these results with previously published data for AFM₁ in milk in Iran shows that the percentage of samples exceeding the EU maximum level is consistently high over the years, indicating a general problem related to AFB₁ contamination in dairy feedingstuff.     

Milk progesterone levels in crossbred buffaloes (swamp x murrah) and a comparison to the original breeds

Progesterone levels in milk fat and whole milk were determined in eight crossbred buffaloes (swamp x murrah). Progesterone peaks in milk fat and whole milk during the luteal phase ranged from 44.23 to 224.50 ng/ml and 8.58 to 20.22 ng/ml, respectively. Progesterone levels were measured in two murrah cows and seven swamp cows for comparison with the levels in the crossbred buffaloes. Variation in milk progesterone levels between breeds was indicated and discussed. Radioimmunoassay for progesterone in milk fat and whole milk of buffaloes was validated.     

Modified Radioimmunoassay Determination for Staphylococcal Enterotoxin B in Foods

The sensitivity of solid-phase radioimmunoassay for the measurement of staphylococcal enterotoxin B (SEB) in foods was decreased by food constituents that react with rabbit anti-SEB with an equivalency of over 2 ng/ml. This activity was minimized by a conditioning step for anti-SEB and by removal of interfering compounds in the sample by extraction. The assay was a sequential solid-phase radioimmunoassay technique in which polystyrene test tubes were initially incubated with antisera and then with bovine serum albumin. The tubes were then conditioned with either a centrifuged aqueous cheese extract or, equally effective, reconstituted nonfat dry milk for 16 h at 4°C. Samples of milk or heat-treated and CHCl₃-extracted cheese or chicken salad slurries were incubated in the assay tubes for 6 h at 37°C. The samples were replaced by ¹²⁵I-labeled SEB and incubated for a further 2 to 4 h before the contents were removed and the tubes were washed and counted. A buffer solution containing known concentrations of toxin served as standards for assaying SEB in the food extracts. The entire assay can be accomplished within 24 h with a sensitivity of 1 ng/ml in milk and in the cheese extract or 1.3 ng/ml in the chicken salad extract.     

The protective effect of breast feeding in relation to sudden infant death syndrome (SIDS): III. Detection of IgA antibodies in human milk that bind to bacterial toxins implicated in SIDS

Two toxin-producing bacteria implicated in sudden infant death syndrome (SIDS) are Staphylococcus aureus and Clostridium perfringens. Epidemiological studies have shown that breast feeding reduces an infant's risk of SIDS. This protective effect could be due partly to IgA antibodies to these toxins in human milk. The aim of this work was to use a quantitative ELISA to determine levels of IgA antibodies that bound to toxic shock syndrome toxin (TSST-1), staphylococcal enterotoxin C (SEC) and C. perfringens enterotoxin A (CEA) in individual samples of human milk. All samples of milk tested contained IgA antibodies that bound to the bacterial toxins. For individual samples, IgA bound to TSST-1, SEC and CEA were in the range of 900-3100 ng ml(-1), 1000-3600 ng ml(-1) and 1000-4300 ng ml(-1) respectively. Isolation of S. aureus from mothers donating breast milk samples was used to determine if the presence of bacteria affected IgA levels which bound TSST-1 and SEC. For 3/5 samples with levels above the upper limit of the standard deviation (2375 ng ml(-1)) for IgA bound to TSST-1, S. aureus was isolated from the mother whilst 4/5 samples found to contain levels above the upper limit of the standard deviation (2627 ng ml(-1)) for IgA bound to SEC, had S. aureus isolated from the mother. In conclusion, if bacterial toxins do play a role in precipitating a SIDS death, the presence of IgA antibodies to toxins in breast milk, but not in infant formula, might contribute to the protective effect of breast feeding in relation to SIDS.     

γ Irradiation of Staphylococcal Enterotoxin B

The inactivation of enterotoxin B by γ irradiation was studied by use of single-and double-gel-diffusion assay techniques. Enterotoxin B (99+% purity) was suspended either in 0.04 m Veronal buffer (pH 7.2) or in milk, dispensed and heat-sealed in borosilicate glass vials, and irradiated essentially at 21 to 26 C with a cobalt-60 source. Parallel titrations of irradiated enterotoxin B in Veronal buffer were made by use of gel-diffusion and cat assay procedures to establish the relative sensitivity of these two assay procedures to irradiated enterotoxin. Results were identical. A dose of 5 Mrad was required to reduce an enterotoxin B concentration of 31 μg/ml in Veronal buffer to less than 0.7 μg/ml. When milk was used as a vehicle, a dose of 20 Mrad was needed to inactivate a 30 μg/ml concentration of enterotoxin B to less than 0.5 μg/ml. With Veronal buffer and milk as vehicles, the D values (dose required to inactivate 90%) for enterotoxin B inactivation were 2.7 and 9.7 Mrad, respectively.     

Detection rate of enterotoxigenic Staphylococcus aureus isolated from children with intestinal disbacteriosis

Detection rate of enterotoxigenic Staphylococcus aureus isolated from faeces of 62 children aged from 3 months old to 7 years old with intestinal dysbacteriosis was studied by indirect hemagglutination assay and enzume immunoassay. It was shown that strains of S.aureus producing staphylococcal enterotoxin A (SEA) are prevailed (40.3%) in children with disturbances of intestinal microflora while staphylococcal enterotoxin B (SEB)-producing strains were detected in 20.9% of children. Amount of produced enterotoxin varied for SEA from 125 ng/ml to 2000 ng/ml and for SEB--from 125 ng/ml to 250 ng/ml. Inverse dependence of detection rate of enterotoxin-producing strains in faeces on age of children was established. The most number of enterotoxigenic strains of S.aureus was detected in infants. These data point to expediency of determination of enterotoxin-producing ability of S. aureus strains isolated from children with dysbacteriosis as measure of danger of this microorganism for children's health and indication for adequate actions for its elimination.     

Establishment of highly specific and quantitative immunoassay systems for staphylococcal enterotoxin A, B, and C using newly-developed monoclonal antibodies

Staphylococcal enterotoxin (SE) activities remain after boiling or treating with proteases. The main symptoms such as vomiting and diarrhea, are caused by the ingestion of SEs. Among SEs, SEA has been reported to be the major and most toxic protein. A highly specific and simple assay system is required to diagnose staphylococcal food poisoning. Therefore, the development of a suitable assay system is strongly anticipated. In this study, we have established a highly specific and sensitive avidin-biotin sandwich ELISA (ABS-ELISA) system for SEA, SEB, and SEC1 using newly-developed monoclonal antibodies. The linearity of these systems obtained was in the range of 0.78-25 ng/ml for each SE, and furthermore, the lower concentrations of SEs could also be detected. The recoveries of SEs from murine serum, skim milk solution, and raw milk were found to be over 90%, suggesting that our systems could detect SEs without any interventions, such as these from milk or serum proteins. We were also able to quantify SEs in 22 specimens of culture supernatants of S. aureus isolated in past occurrences. Our established system should be very useful not only in the clinical field but also in various fields of investigation because of its quantifi-cation and simplicity in detecting SEs.     

Direct radioimmunoassay of progesterone in milk using a radioiodinated progesterone derivative

A heterologous radioimmunoassay technique which allows the measurement of progesterone in whole milk, without extraction is described. An antiserum against progesterone-12α-succinyl-BSA and progesterone-11α-(2′-methyl)succinyl-¹²⁵I-thyramine was used. When applied in pregnancy detection on day 20–22, progesterone levels of up to 6 ng/ml or above 8 ng/ml were indicative of an unsuccessful or a successful insemination, respectively.The sensitivity of the assay was 0.8 ng/ml, and the values found in cow milk ranged from 0.8 to 50 ng/ml.     

Milk fat progesterone concentrations in goats and early pregnancy diagnosis

Blood and milk samples from foremilk during afternoon milking, were simultaneously collected from 285 dairy goats. In experiment 1, fiva cyclic goats were sampled daily for 21 days. In experiment 2, 280 females from 9 flocks were submitted to sampling 21 days after insemination. In addition, some milk samples were divided in two parts, after which one was frozen and the other kept at +4 degrees C until assay. Progesterone concentrations were measured in blood, whole milk and milk fat by radioimmunoassay. No difference in whole milk or fat progesterone levels was found between frozen and refrigerated milk samples. Milk butterfat progesterone concentrations paralleled those in plasma or whole milk throughout the estrous cycle and ranged from about 20 ng/ml at estrus to about 400 ng/ml in mid-luteal phase. The ratio of mid-luteal phase progesterone levels to those seen in the estrous period was over 20 in fat and in blood. This ratio was very much lower in whole milk. Consequently the determination of pregnant and non-pregnant goats from the samples collected 21 days after service was very much easier and accuracy was better when the progesterone content was assayed from milk fat than from whole milk. It was concluded that early pregnancy diagnosis in goats can be done routinely by determination of progesterone levels in milk fat.     

Detection and production of verotoxin 1 of Escherichia coli O157:H7 in food.

Verotoxin 1 (VT1) is a recognized virulence factor of Escherichia coli O157:H7, a cause of severe food-borne disease. The public health significance of preformed verotoxin in food is unknown, and relatively little research has been done to determine the production of VT1 in food. The purposes of this study were to develop a sensitive method to detect VT1 in milk and in ground beef and to determine the conditions for VT1 production in these foods. A sandwich enzyme-linked immunosorbent assay in which we used VT1-specific monoclonal antibody 9C9F5 as the capture antibody and a rabbit polyclonal antibody raised against VT2 as the detection antibody was developed for the detection and quantification of VT1 in milk and in ground beef. The enzyme-linked immunosorbent assay was sensitive to a minimum of 0.5 ng of VT1 per ml of milk and 1.0 ng of VT1 per g of ground beef. The greatest amount of VT1 detected in milk (306 ng/ml) was detected in samples that were incubated at 37 degrees C with agitation (160 rpm) for 48 h. Very little toxin (1 ng/ml) was produced at 25 or 30 degrees C within 96 h. VT1 production was greater in ground beef than in milk; 452 ng of VT1 per g was produced in beef at 37 degrees C in 48 h. Relatively little VT1 was produced in beef within 96 h at 25 and 30 degrees C (2.1 and 9.8 ng of VT1 per g, respectively). Our results indicate that ground beef is a better medium for VT1 production than milk.(ABSTRACT TRUNCATED AT 250 WORDS)     

Detection and production of verotoxin 1 of Escherichia coli O157:H7 in food.

Verotoxin 1 (VT1) is a recognized virulence factor of Escherichia coli O157:H7, a cause of severe food-borne disease. The public health significance of preformed verotoxin in food is unknown, and relatively little research has been done to determine the production of VT1 in food. The purposes of this study were to develop a sensitive method to detect VT1 in milk and in ground beef and to determine the conditions for VT1 production in these foods. A sandwich enzyme-linked immunosorbent assay in which we used VT1-specific monoclonal antibody 9C9F5 as the capture antibody and a rabbit polyclonal antibody raised against VT2 as the detection antibody was developed for the detection and quantification of VT1 in milk and in ground beef. The enzyme-linked immunosorbent assay was sensitive to a minimum of 0.5 ng of VT1 per ml of milk and 1.0 ng of VT1 per g of ground beef. The greatest amount of VT1 detected in milk (306 ng/ml) was detected in samples that were incubated at 37 degrees C with agitation (160 rpm) for 48 h. Very little toxin (1 ng/ml) was produced at 25 or 30 degrees C within 96 h. VT1 production was greater in ground beef than in milk; 452 ng of VT1 per g was produced in beef at 37 degrees C in 48 h. Relatively little VT1 was produced in beef within 96 h at 25 and 30 degrees C (2.1 and 9.8 ng of VT1 per g, respectively). Our results indicate that ground beef is a better medium for VT1 production than milk.(ABSTRACT TRUNCATED AT 250 WORDS)     

Growth and enterotoxin production of Staphylococcus aureus during the manufacture and ripening of Camembert-type cheeses from raw goats' milk

Tests were carried out to determine the effect of manufacturing procedures for a Camembert-type cheese from raw goats' milk on the growth and survival of Staphylococcus aureus organisms added to milk at the start of the process, and to study the possible presence of staphylococcal enterotoxin A in these cheeses. The initial staphylococcal counts were, respectively, 2, 3, 4, 5 and 6 log cfu ml⁻¹. Cheese was prepared following the industrial specifications and ripened for 41 d. Detection of enterotoxins was done by the Vidas SET test and by an indirect double-sandwich ELISA technique using antienterotoxin monoclonal antibodies. Generally, numbers of microbes increased at a similar rate during manufacture in all cheeses until salting. During the ripening period, the aerobic plate count population and Staph. aureus levels remained stable and high. There was an approximately 1 log reduction of Staph. aureus in cheeses made with an initial inoculum of Staph. aureus greater than 10³ cfu ml⁻¹ at the end of the ripening period (41 d) compared with the count at 22 h. The level of staphylococcal enterotoxin A recovered varied from 1 to 3·2 ng g⁻¹ of cheese made with an initial population of 10³–10⁶ cfu ml⁻¹. No trace of enterotoxin A was detected in cheeses made with the lowest Staph. aureus inoculum used in this study.     

Development of a sandwich ELISA assay for measuring bovine soluble type II IL-1 receptor (IL1R2) concentration in serum and milk

The IL1R is composed of two kinds of molecule, type I (IL1R I) and type II (IL1R2). IL1R1 contributes to IL-1 signaling, whereas the IL1R2 has no signaling property and acts as a decoy for IL-1. In this study, we developed a bovine IL1R2-specific sandwich ELISA to examine the sIL1R2 concentration in serum and milk from dairy cows. The concentration of colostral IL-1beta was examined to estimate the correlation to sIL1R2. The results showed that the sIL1R2 concentration in sera and milk changes with the stages of lactation. The serum sIL1R2 concentrations were 5.56+/-0.69 ng/ml (colostrum), 3.14+/-0.72 ng/ml (the early stage of lactation) and 5.76+/-1.25 ng/ml (the late stage of lactation). The milk sIL1R2 concentrations were 1.83+/-0.47 ng/ml (colostrum), 0.73+/-0.22 ng/ml (the early stage of lactation) and 2.92+/-0.56 ng/ml (the late stage of lactation). The concentrations of IL1R2 in sera and milk were significantly higher at the late stage of lactation and colostrum than that of the early stage of lactation. The reduction rates of sIL1R2 levels from the colostrum to the early stage of lactation were 43.6% (serum) and 61% (whey). IL-1beta was detected in all the colostrum (995.9+/-346.6 ng/ml). Significant correlation was observed between the levels of colostral IL-1beta and IL1R2 (r=0.75).     

Epidermal growth factor-like proteins in breast fluid and human milk

Epidermal growth factor (EGF), and the transforming growth factor-alpha (TGF-alpha) family of proteins, which also bind to the EGF receptor, have been associated with human breast cancer. The total EGF-like proteins were determined by a radioreceptor assay, and TGF-alpha by radioimmunoassay, in human milk and breast fluid samples. The breast fluids were collected by nipple aspiration from healthy premenopausal women. Both the 24 milks and 18 breast fluids assayed contained EGF-like proteins, at concentrations ranging from 32-600 ng/ml (median, 140 ng/ml), and 62-654 ng/ml (median, 205 ng/ml) respectively. Immunoreactive TGF-alpha proteins were detected at higher levels in 21 breast fluids (range, 0-50.0; median 5.1 ng/ml) than in 24 milk samples (range, 0-8.4; median, 0.8 ng/ml).     

Production of staphylococcal enterotoxins C1 and C2 and thermonuclease throughout the growth cycle

Synthesis of enterotoxins C1 and C2 and thermonuclease throughout the growth cycle was investigated with Staphylococcus aureus type strains FRI137 and FRI361 and S. aureus isolates M5 (C1) and L2 (C2) of animal origin. Both enterotoxins were produced during the exponential growth phase or at the beginning of the stationary phase. The minimal incubation time (7 to 12 h) and the lowest population (10(7) to 2 x 10(9) CFU/ml) associated with detectable enterotoxin (1 to 6.5 ng/ml) were related to the total amount of toxin produced after 24 h. Thermonuclease was detected in all samples whenever enterotoxins were detected. Furthermore, strain FRI137 produced thermonuclease earlier and at lower cell populations than it did enterotoxin C1. Patterns of enterotoxin and thermonuclease synthesis did not correlate. The concentration of toxins increased throughout the growth cycle, while the concentration of thermonuclease remained constant during the last hours of the growth cycle.     

Aflatoxin M1 contamination in commercial pasteurized milk from local markets in Fariman, Iran

Contamination of milk and dairy products with aflatoxin M₁ (AFM₁) presents a risk for human health. The aim of this study was to investigate the presence of AFM₁ in pasteurized milk samples in Fariman, located in the province of Khorasan Razavi, Iran, by enzyme-linked immunosorbent assay (ELISA). Forty-five samples of pasteurized milk from different supermarkets were collected during 3 months in summer (July to September, 2012). AFM₁ contamination was detected in all of milk samples. The mean concentration of aflatoxin M₁ was 27.2 ng/l. The range of AFM₁ content was 8.8–64 ng/l. Thirteen (28.8 %) of the samples had AFM₁ levels exceeding the maximum levels (50 ng/l) accepted by the European Union. Due to the fact that milk is used by all the age groups including infants and children in Fariman city, it is necessary to minimize the health risk from AFM1 contamination in milk. For this reason, the level of its precursor, aflatoxin B₁ (AFB₁), in dairy feeds must be reduced, requiring constant aflatoxin monitoring of relevant agricultural commodities.