SEA), B Research Articles

Development of a novel multiplex electrochemiluminescent-based immunoassay for quantification of human serum IgG against 10 Staphylococcus aureus toxins

An electrochemiluminescent (ECL)-based multiplex immunoassay using Meso-Scale Discovery (MSD) technology was developed for detecting antibody response toward 10 Staphylococcus aureus (S. aureus) exotoxins. These 10 antigens included three different groups of toxins: 1) single component pore-forming toxins such as alpha- and delta-hemolysins, 2) the bicomponent pore-forming toxin Panton–Valentine leukocidin (PVL), comprised of LukS-PV and LukF-PV subunits, and 3) enterotoxin/superantigens — Staphylococcal enterotoxins A (SEA), B (SEB), C1 (SEC1), D (SED), K (SEK) and Toxic shock syndrome toxin-1 (TSST-1). Assay development included optimization steps with a conventional SEB ELISA-based serological assay and then optimized parameters were transferred and re-optimized in a singleplex ECL format. Finally, two pentaplex solid-phase ECL formats were developed. As proof of concept, one set of pentaplex ECL data was compared with conventional ELISA results. During the assay development controls were screened and developed for both the singleplex and multiplex assays. ECL-based multiplex assays were more sensitive with a wide dynamic range and proved more time-efficient than conventional ELISAs. Using the newly developed ECL method we showed, for the first time, that delta-hemolysin toxin can induce an immune response as antibody titers could be detected.

Heterologous expression of staphylococcal enterotoxin B (seb) gene for antibody production

Staphylococcal food poisoning (SFP) is caused by the members of superantigen family called staphylococcal enterotoxins (SEs). About 20 different types of SEs are produced by Staphylococcus aureus out of which type A (SEA), B (SEB), C (SEC) and D (SED) are commonly implicated in SFP. Among these, SEB is the most potent toxin and has also gained the status of biological warfare (BW) agent. Therefore, detection of SEB is of utmost importance. Any immunological detection system for SEB requires specific and sensitive antibodies which inturn depends on the purity of the SEB. In the present investigation, seb gene of S. aureus was cloned and expressed in E. coli along with biotin as fusion partner to facilitate the purification process. The yield of purified recombinant SEB was 13.1 mg/L of culture broth. Biotin tag from the biotinylated toxin was removed by protease cleavage, and both biotinylated and non-biotinylated toxin types were used for raising hyperimmune antiserum. Antisera were also specific for SEB amongst different kinds of food poisoning agents tested by indirect plate ELISA and western blot analysis. The quality of the antisera raised in this study was found superior to the commercially available antiserum. The investigation suggests that construction of recombinant staphylococcal enterotoxin B is a good alternative for production of pure enterotoxin to be used in antibody generation.

176 EFFECT OF LEUKEMIA INHIBITORY FACTOR FROM HUMAN AND MOUSE ORIGIN ON THE DEVELOPMENT OF BOVINE EMBRYOS PRODUCED IN VITRO

Leukemia Inhibitory Factor (LIF) is a cytokine with potential to influence embryonic quality and proliferation within the inner cell mass (ICM). However, conflicting effects of LIF have been reported with in vitro-produced (IVP) bovine embryos, in spite of LIF receptor (LIFr) and gp130 transcripts being expressed at all stages during pre-implantation development (Niemann and Wrenzycki 2000 Theriogenology 53, 21–34). As there is no commercially available bovine LIF (bLIF), researchers have used human LIF (hLIF) because of its greater sequence homology compared to murine LIF (mLIF). However, mLIF has been not compared with hLIF in culture with bovine embryos; thus this was the aim of this study. Cumulus–oocyte complexes from slaughterhouse ovaries were matured and fertilized in vitro and presumptive zygotes cultured in modified synthetic oviduct fluid with 6 g L-1 BSA. At 139 h post-insemination (Day 6), a total of 423 morulae (>90%) and early blastocysts were cultured for 48 h with: (1) 100 ng mL-1 recombinant mLIF (Sigma-Aldrich Quimica SA, Madrid, Spain); (2) 100 ng mL-1 recombinant hLIF (Sigma); and (3) no LIF. Data (6 replicates) were processed by GLM and Duncan's test, and expressed as LSM � SE (ab: P < 0.05; xy: P < 0.01). Development was recorded up to the hatched blastocyst stage and cells were differentially counted in the ICM and trophectoderm (TE) following the method described by Thouas et al. (2001 Reprod. Biomed. Online 3, 25–29). There were no differences within developmental rate on Day 7, but reduced blastocyst rates were observed on Day 8 between hLIF (42.0 � 3.9a and 27.2 � 3.3a) and controls (57.7 � 3.9b and 38.9 � 3.3b) at the medium and expanded stages, respectively, whereas mLIF had no effect (47.4 � 3.9 and 32.3 � 3.3). Contrary to development, Day 8 blastocysts showed decreased cell counts in both the ICM and the ICM/total cell proportions in the presence of mLIF (19.1 � 3.1x and 13.8 � 2.4x vs. 32.6 � 3.0y and 24.8 � 2.3y for controls, respectively), whereas hLIF had no effect (29.7 � 3.1y and 20.9 � 2.4y). No changes were seen in TE and total cell counts. The disparate effects exhibited by hLIF and mLIF during blastocyst formation may reflect the fact that these compounds are inappropriate to replace bLIF, and/or endogenous LIF probably suffices during bovine development. In fact, mouse embryonic development and blastocyst cell numbers decrease in murine embryos injected with LIF antisense nucleotides (Cheng et al. 2004 Biol. Reprod. 70, 1270–1276). Furthermore, embryonic stem (ES)-like cell derivation in bovine is possible with (Saito et al. 2003 Biochem. Biophys. Res. Com. 309, 104–113) and without (Mitalipova et al. 2001 Cloning 3, 59–67) exogenous LIF. Therefore, strategies to investigate LIF signalling in bovine embryos and stem cells should be reconsidered. This work was supported by Grant AGL2005-04479.

The protective effect of immunisation against diphtheria, pertussis and tetanus (DPT) in relation to sudden infant death syndrome.

Epidemiological evidence indicates infants immunised against diphtheria, pertussis and tetanus (DPT) are at decreased risk of sudden infant death syndrome (SIDS). Asymptomatic whooping cough and pyrogenic toxins of Staphylococcus aureus have been implicated in the aetiology of SIDS. The objectives of the present study were: (1) to determine if the DPT vaccine induced antibodies cross-reactive with the staphylococcal toxins; (2) to determine if antibodies to the pertussis toxin (PT) and the staphylococcal toxins were present in the sera of women during late pregnancy; (3) to examine the effects of infant immunisation on levels of antibodies to PT and the staphylococcal toxins; (4) to assess the effects of changes in immunisation schedules in the UK on the incidence and age distribution of SIDS. Enzyme-linked immunosorbent assays (ELISA) were used to measure binding of rabbit or human IgG to the DPT vaccine, PT, toxic shock syndrome toxin-1 (TSST-1) and staphylococcal enterotoxins A (SEA), B (SEB) and C (SEC). Neutralisation activity of anti-DPT serum was assessed by a bioassay for induction of nitric oxide from human monocytes by the staphylococcal toxins. Anti-DPT serum bound to the DPT vaccine, PT and each of the staphylococcal toxins. It also reduced the ability of the four toxins to induce nitric oxide from monocytes. In pregnant women, levels of IgG to PT, SEC and TSST-1 decreased significantly in relation to increasing weeks of gestation while antibodies to SEA and SEB increased. In infants' sera there were significant correlations between levels of IgG bound to DPT and IgG bound to PT, TSST-1 and SEC but not SEA or SEB. Antibody levels to the toxins in infants declined with age; sera from infants < or = 2 months of age had higher levels of IgG bound to the toxins than those older than 2 months. This pattern was observed for infants whose immunisation schedules began at 2 months of age or 3 months of age. The decrease in IgG bound to the toxins was, however, less for those immunised at 2 months. The decrease in SIDS deaths after the change in immunisation schedules was greatest in the 4-6-month age range. While DPT immunisation might prevent some unexplained infant deaths due to asymptomatic whooping cough, these data indicate that immunisation with DPT also induces antibodies cross-reactive with pyrogenic staphylococcal toxins implicated in many cases of SIDS. Passive immunisation of infants who have low levels of these antibodies might reduce further the numbers of these infant deaths.

The protective effect of immunisation against diphtheria, pertussis and tetanus (DPT) in relation to sudden infant death syndrome

Epidemiological evidence indicates infants immunised against diphtheria, pertussis and tetanus (DPT) are at decreased risk of sudden infant death syndrome (SIDS). Asymptomatic whooping cough and pyrogenic toxins of Staphylococcus aureus have been implicated in the aetiology of SIDS. The objectives of the present study were: (1) to determine if the DPT vaccine induced antibodies cross-reactive with the staphylococcal toxins; (2) to determine if antibodies to the pertussis toxin (PT) and the staphylococcal toxins were present in the sera of women during late pregnancy; (3) to examine the effects of infant immunisation on levels of antibodies to PT and the staphylococcal toxins; (4) to assess the effects of changes in immunisation schedules in the UK on the incidence and age distribution of SIDS. Enzyme-linked immunosorbent assays (ELISA) were used to measure binding of rabbit or human IgG to the DPT vaccine, PT, toxic shock syndrome toxin-1 (TSST-1) and staphylococcal enterotoxins A (SEA), B (SEB) and C (SEC). Neutralisation activity of anti-DPT serum was assessed by a bioassay for induction of nitric oxide from human monocytes by the staphylococcal toxins. Anti-DPT serum bound to the DPT vaccine, PT and each of the staphylococcal toxins. It also reduced the ability of the four toxins to induce nitric oxide from monocytes. In pregnant women, levels of IgG to PT, SEC and TSST-1 decreased significantly in relation to increasing weeks of gestation while antibodies to SEA and SEB increased. In infants’ sera there were significant correlations between levels of IgG bound to DPT and IgG bound to PT, TSST-1 and SEC but not SEA or SEB. Antibody levels to the toxins in infants declined with age; sera from infants ≤2 months of age had higher levels of IgG bound to the toxins than those older than 2 months. This pattern was observed for infants whose immunisation schedules began at 2 months of age or 3 months of age. The decrease in IgG bound to the toxins was, however, less for those immunised at 2 months. The decrease in SIDS deaths after the change in immunisation schedules was greatest in the 4–6-month age range. While DPT immunisation might prevent some unexplained infant deaths due to asymptomatic whooping cough, these data indicate that immunisation with DPT also induces antibodies cross-reactive with pyrogenic staphylococcal toxins implicated in many cases of SIDS. Passive immunisation of infants who have low levels of these antibodies might reduce further the numbers of these infant deaths.

Detection of pyrogenic toxins of Staphylococcus aureus in sudden infant death syndrome.

It has been suggested that pyrogenic toxins of Staphylococcus aureus are involved in the series of events leading to some cases of sudden infant death syndrome (SIDS). The objectives of the study were to screen tissues from SIDS infants for pyrogenic toxins and to compare incidence of identification of these toxins among these infants from different countries. An enzyme-linked immunosorbent assay (ELISA) and a flow cytometry method were used to screen body fluids and frozen or formalin-fixed tissues for pyrogenic toxins of S. aureus, toxic shock syndrome toxin 1 (TSST), staphylococcal enterotoxins A (SEA), B (SEB), and C1 (SEC). Toxins were identified in tissues of 33/62 (53%) SIDS infants from three different countries: Scotland (10/ 19, 56%); France (7/13, 55%); Australia (16/30, 53%). In the Australian series, toxins were identified in only 3/19 (16%) non-SIDS deaths (chi2 = 5.42, P < 0.02). The flow cytometry method was useful for toxin detection in both frozen and fixed tissues, but ELISA was suitable only for frozen tissues or those fixed for less than 12 months. Identification of pyrogenic toxins in > 50% of SIDS infants from three different countries indicated further investigation into the role the toxins play in cot deaths might result in development of additional measures to reduce further the incidence of these infant deaths.

Detection of pyrogenic toxins of Staphylococcus aureus in sudden infant death syndrome

It has been suggested that pyrogenic toxins of Staphylococcus aureus are involved in the series of events leading to some cases of sudden infant death syndrome (SIDS). The objectives of the study were to screen tissues from SIDS infants for pyrogenic toxins and to compare incidence of identification of these toxins among these infants from different countries. An enzyme-linked immunosorbent assay (ELISA) and a flow cytometry method were used to screen body fluids and frozen or formalin-fixed tissues for pyrogenic toxins of S. aureus, toxic shock syndrome toxin 1 (TSST), staphylococcal enterotoxins A (SEA), B (SEB), and C 1 (SEC). Toxins were identified in tissues of 33/62 (53%) SIDS infants from three different countries: Scotland (10/19, 56%); France (7/13, 55%); Australia (16/30, 53%). In the Australian series, toxins were identified in only 3/19 (16%) non-SIDS deaths (χ 2=5.42, P<0.02). The flow cytometry method was useful for toxin detection in both frozen and fixed tissues, but ELISA was suitable only for frozen tissues or those fixed for less than 12 months. Identification of pyrogenic toxins in >50% of SIDS infants from three different countries indicated further investigation into the role the toxins play in cot deaths might result in development of additional measures to reduce further the incidence of these infant deaths.

Characteristics of Staphylococcus aureus strains isolated from clinical and non-clinical human sources in Trinidad: Susceptibility to bacteriophages and antimicrobial agents, and toxigenicity

The susceptibility of Staphylococcus aureus strains isolated from human clinical and non-clinical sources in Trinidad to bacteriophages and antimicrobial agents was determined. The ability of the strains to produce enterotoxins and toxic shock syndrome toxin-1 (TSST-1) was also investigated. Of the 554 strains tested, 454 (81.8%) were susceptible to international phage set (IPS) phages with strains isolated from bacteruria (57.1%) and bacteremia (53.3%) having a low sensitivity compared to isolates from aspirates (87.3%) and anterior nares (97.4%). All sources combined, strains were most susceptible to phages belonging to several groups (mixed). Overall, 419 (75.6%) strains were resistant to one or more of nine antimicrobial agents tested. Resistance to penicillin was most prevalent, with 413 (74.5%) strains found to be resistant. Prevalence of resistance to tetracycline, gentamicin, oxacillin, cefuroxime and ciprofloxacin was 5.1%, 2.0%, 0.7%, 0.4% and 0.4%, respectively. Of the 554 strains tested, 307 (55.4%) produced staphylococcal enterotoxins A (SEA), B (SEB), C (SEC) and D (SED) singly or in combination. Strains recovered from high vaginal swabs were least enterotoxigenic (40.0%) as compared to umbilical infection isolates which were most enterotoxigenic (78.9%). TSST-1 was produced by 95 (19.0%) out of 499 strains tested, with isolates from bacteruria found to be most toxigenic (33.3%). It was concluded that the S. aureus strains tested were highly susceptible to bacteriophages and antimicrobial agents (except penicillin) and that enterotoxigenic and TSST-1 producers were widespread and have an aetiologic potential.

The absence of evidence of staphylococcal toxin involvement in the pathogenesis of Kawasaki disease.

To detect a causative superantigen and to clarify a possible role for staphylococci in Kawasaki disease (KD), culture supernatants of individual bacterial isolates from 11 acute-stage patients were studied. Toxic shock syndrome toxin-1 (TSST-1) and antibody to TSST-1 and enterotoxins A (SEA), B (SEB), and C (SEC) in acute (mean, day 7) and late convalescent (mean, month 15) sera from 26 patients (12 with coronary artery aneurysms) and 22 age-matched controls were measured. Only 1 of 60 supernatants was mitogenic for human lymphocytes; it was 1 of the 4 Staphylococcus aureus isolates. Mitogenicity was neutralized by sera obtained after administration of intravenous gamma globulin (mean, week 4) but not by late convalescent sera. TSST-1 was detectable in 2 of 26 acute sera and 1 of 22 control sera. No KD but 1 control serum had IgM to TSST-1. IgG seroconversion rates to TSST-1, SEA, SEB, and SEC were 10%, 15%, 21% and 16%, respectively. These data do not support the involvement of toxin-producing staphylococci in KD.

Bacteriologic Quality of Some Trinidadian Ready-to-Consume Foods and Drinks and Possible Health Risks to Consumers

The bacteriologic quality of 10 ready-to-consume Trinidadian foods and drinks was determined by culturing for Staphylococcus aureus, Escherichia coli, Salmonella spp. and thermophilic Campylobacter spp. The toxigenicity of S. aureus and E. coli strains was determined and the susceptibility of S. aureus isolates to bacteriophages was also investigated. Of a total of 293 food samples tested, 58 (19.8%), 30 (10.2%), and 10 (3.4%) were positive for S. aureus, E. coli, and Salmonella spp., respectively. All samples were negative for Campylobacter but only two food types, chutney and preserved fruits, were negative in all pathogen assays. The sausage known as black pudding (boudin noir) was significantly (P ≤ 0.001; χ2 more contaminated with S. aureus (73.0%) than other foods and was also significantly (P ≤ 0.0l; χ2) more contaminated (43.2%) than all other foods except seamoss (33.3%) and roti (20.0%), where the difference was not significant (P ≥ 0.05; χ2). Black pudding also yielded all 10 strains of Salmonella isolated; the prevalent serotypes were S. agona (4 isolates) and S. derby (3 isolates). All 30 isolates of E. coli were negative for heat-labile enterotoxins (LT) and verotoxins (VT). Of the 58 S. aureus isolates, 33 (56.9%) were susceptible to bacteriophages with 19 (70.4%) of 20 black-pudding and 12 (66.7%) of 18 ice-cream isolates typeable. None of the isolates recovered from local sweets, snow cones, and roti was typeable. Regardless of the food source, strains of S. aureus were most susceptible to phages in various groups (mixed) constituting 21 (63.6%) of 33 strains typeable, followed by phages in groups III (18.2%) and V (15.2%). Twenty-two (37.9%) strains of S. aureus were enterotoxigenic with 8 (13.8%), 5 (8.6%), 13 (22.4%), and 12 (20.7%) strains producing, either singly or mixed, Staphylococcal enterotoxins A(SEA), B(SEB), C(SEC), and D(SED) respectively. It was concluded that black pudding, a local delicacy, poses the greatest health risk to consumers, making it imperative to institute sanitary measures during its production and sale.

Characteristics of Staphylococcus aureus strains isolated from bovine mastitic milk: bacteriophage and antimicrobial agent susceptibility, and enterotoxigenicity.

Staphylococcus aureus strains isolated from bovine mastitic milk in Trinidad were examined for their susceptibility to bacteriophages and antimicrobial agents and their ability to produce enterotoxins. Phage 42D was used to screen for bovine strains of S. aureus in milk. Of 250 strains tested, 224 (89.6%) were sensitive to phages in the international phage set (IPS), 85 (34.0%) were resistant to antimicrobial agents and 134 (53.6%) were enterotoxigenic. Strains lysed by phages in various groups (mixed) were prevalent, 145 (58.0%), followed by strains sensitive to groups III (17.0%) and I (8.8%) phages. A total of 72 (28.8%) strains were lysed by phage 42D either alone or with others. Resistance to penicillin was most common with 59 (23.6%) strains while 44 (17.6%) and 43 (17.2%) strains were resistant to ampicillin and triple sulphur respectively. Only 3 (1.2%) strains were resistant to methicillin. Prevalence of resistance to penicillin (12.5%) amongst phage 42D-sensitive strains was significantly (P < or = 0.01; X2) lower than for strains not lysed by phage 42D (28.1%) but strains susceptible to phage 42D were significantly (P < or = 0.05; X2) more resistant (4.2%) to methicillin than those not lysed by the phage (0.0%). Amongst 134 enterotoxigenic strains, 32 (23.9%), 77 (57.5%), 67 (50.0%) and 21 (15.7%) produced staphylococcal enterotoxins A(SEA), B(SEB), C(SEC) and D(SED) respectively either alone or mixed. SEB and SEC were significantly (P < or = 0.01; X2) more produced than either SEA or SED. Strains lysed by groups IV, i.e. 42D (62.5%), and III (56.7%) were more enterotoxigenic than those sensitive to phages in groups II (45.5%) and non-typable (46.2%) but the differences were not statistically significant (P > or = 0.05; X2). Strains lysed by group II phages (72.7%) were significantly (P < or = 0.05; X2) more resistant to antimicrobial agents than those lysed by phage 42D (18.8%). It was concluded that bovine mastitis strains of S. aureus in Trinidad were highly susceptible to bacteriophages and antimicrobial agents and enterotoxigenic and less than one-third may be considered to be bovine strains.

Bacteriological quality and associated public health risk of pre-processed bovine milk in Trinidad

The bacteriological quality of pre-processed raw milk originating from all 16 milk collection centres in Trinidad was evaluated. The mean total aerobic counts for bacteria, Staphylococcus aureus and Escherichia coli were determined. The pH and presence of somatic cells in milk were also determined. Of the 507 milk samples collected, 454 (89.5%) were California mastitis test (CMT)-positive and the mean pH was 6.50±0.13. The total aerobic plate count per ml was generally high for all samples ranging from 5.8 × 10 5 ±3.1 × 10 5 to 5.7 × 10 8 ± 1.5 × 10 9. S. aureus was isolated from 478 (94.3%) samples and the mean counts per ml ranged from 1.5 × 10 5 ± 1.1 × 10 5 to 9.4 × 10 5 ± 1.3 × 10 6. Nine (7.7%) of 117 strains of S. aureus produced staphylococcal enterotoxins A(SEA), B(SEB), D(SED) or a combination. E. coli was isolated from 105 (20.7%) samples and the mean counts per ml ranged from 6.6 × 10 2 ± 1.1 × 10 2 to 4.0 × 10 5 ± 4.6 × 10 5. Twenty-five (23.6%) of the 106 strains of E. coli tested produced verocytotoxin (VT) but none was positive for heat-labile (LT) toxin. It was concluded that pre-processed milk in Trinidad is of poor bacteriological quality. The detection of high counts of S. aureus in milk with some producing heat stable enterotoxins, coupled with the isolation of some verocytotoxigenic E. coli, are of public health significance to consumers.

In vivo antitumor efficacy of tumor-draining lymph node cells activated with nonspecific T-cell reagents.

Progressive growth of immunogenic murine tumors elicits a tumor-specific but functionally deficient T-cell immune response in the draining lymph nodes. These T cells, referred to as "pre-effector" cells could be induced in vitro to differentiate into mature immune effector cells, capable of mediating the regression of established metastases. Initially, tumor cells were used to stimulate the in vitro maturation of pre-effector cells. Alternatively, we found that pre-effector cells could be activated by sequential stimulation with anti-CD3 and interleukin-2 in the absence of tumor cells. In adoptive immunotherapy, these activated cells mediated therapeutic effects that were exquisitely specific to the tumor that triggered the pre-effector cell response in vivo. Since the anti-CD3 interaction with T cells is polyclonal, the activated lymph node cell population must also contain a significant number of T cells that do not have tumor specificity. In an attempt to selectively activate tumor-sensitized pre-effector cells, we recently utilized superantigenic bacterial toxins as T-cell stimuli for effector cell generation. Superantigens combine with major histocompatibility class II molecules to form the ligands that stimulate T cells bearing distinct T-cell receptor V beta elements. Lymph node cells draining the MCA 205 sarcoma stimulated with staphylococcal enterotoxins A (SEA), B (SEB), or C2 (SEC2) resulted in selective expansions of V beta 3 and 11, V beta 3 and 8, or V beta 8.2 T cells, respectively. Adoptive immunotherapy experiments revealed that SEB- and SEC2-, but not SEA- stimulated cells, mediated tumor-specific eradication of pulmonary metastases.(ABSTRACT TRUNCATED AT 250 WORDS)

Phage susceptibility, enterotoxigenicity and antibiograms of staphylococcus aureus strains isolated from human wounds and diarrhoea

The phage types, enterotoxigenicity and antibiograms of Staphylococcus aureus strains isolated from human diarrhoea and skin wounds in Nigeria were determined. Of 194 strains tested, 140 (72.2%) were typable using a combination of phages in the International Phage Set (IPS) for human strains of S. aureus and the bovine phage set. IPS phages lysed 118 (60.8%) strains while 96 (49.5%) were susceptible to bovine phages. S. aureus strains from adult diarrhoea were significantly (P less than or equal to 0.01; x2) more sensitive to bovine phages (52.8%) than to IPS human phages (16.7%). Strains isolated from wounds were however significantly (P less than or equal to 0.001; x2) more susceptible to IPS human phages (72.9%) than to bovine phages (41.5%). Phage group III strains were predominant amongst diarrhoeal isolates while wound strains were most susceptible to group II phages. Phage 119, a bovine phage, lysed all 19 strains of diarrhoeal origin sensitive to group M phages but all wound strains were resistant. Overall, 132 (68.0%) strains were enterotoxigenic producing staphylococcal enterotoxins A (SEA), B (SEB), C (SEC) or a combination of these. A majority of diarrhoeal strains elaborated SEC while SEB production was predominant amongst wound strains. Of the eight antimicrobial agents used, S. aureus strains were most resistant to penicillin (71.6%) and least to sulfamethoxazole/trimethoprim (1.0%). Occurrence of resistance to one or more antibiotics was higher amongst wound strains (97.5%) than amongst strains isolated from diarrhoea (52.6%). It was concluded that bovine phage 119 may be a useful epidemiologic marker for S. aureus strains of bovine origin associated with human diarrhoea in Nigeria. It is however difficult to ascribe any aetiological significance to these strains because other enteropathogens not assayed for may have been present.

Large-scale purification of staphylococcal enterotoxins A, B, and C2 by dye ligand affinity chromatography

A simple method for the purification of staphylococcal enterotoxins A (SEA), B (SEB), and C2 (SEC2) from fermentor-grown cultures was developed. The toxins were purified by pseudo-affinity chromatography by using the triazine textile dye "Red A" and gave overall yields of 49% (SEA), 44% (SEB), and 53% (SEC2). The purified toxins were homogeneous when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but isoelectric focusing of the preparations revealed the microheterogeneity associated with these toxins. The SEA and SEB preparations each consisted of two isoelectric forms with pI values of 7.3 and 6.8 (SEA) and 8.9 and 8.55 (SEB); in contrast, SEC2 contained five different isoelectric forms, with pI values ranging between 7.6 and 6.85. The pattern of elution of the isoelectric forms from the column indicated a cationic-exchange process involved in the binding of toxin to Red A. Such a method forms the basis of a high-yielding, rapid means of purifying the staphylococcal enterotoxins that can easily be adapted to large-scale production.

Analytical methods for Staphylococcus aureus

The staphylococcal enterotoxins are relatively low molecular weight proteins, 27000 to 29000 Da (Bergdoll, 1979). They are produced by the staphylococci, primarily Staphylococcus aureus, although the newer species, Staphylococcus intermedius and Staphylococcus hyicus have been reported to be enterotoxigenic (Adesiyun et al., 1984; Fukuda et al., 1984). The guideline followed for many years in the Food Research Institute to determine whether staphylococci might produce enterotoxin was to determine whether the particular strain produced either coagulase or thermonuclease (TNase). Formerly, both S. intermedius and S. hyicus would have been classified as S. aureus. Therefore, if we follow the current species classification, we cannot confine our testing of staphylococcal strains to S. aureus alone. To my knowledge, no one is currently working on the identification of additional enterotoxins although we do know that unidentified ones do exist. This is very difficult to accomplish because a specific biological test such as the monkey feeding test is necessary to identify the enterotoxin (Surgalla et al., 1953). Apparently, only about five percent of staphylococcal food poisoning outbreaks are due to unidentified enterotoxins. The enterotoxins that have been identified so far are classified as enterotoxins A (SEA), B (SEB), C 1 (SEC1), C~ (SEC2), C 3 (SEC3), D (SED) and E (SEE). The enterotoxin Cs are very closely related and can be identified by their cross-reactions with antibodies prepared against any one of the SECs. The other enterotoxins are identified by antibodies specific for each of the enterotoxins, although cross-reactions between SEB and the SECs (Lee et al., 1980) and SEA and SEE (Lee et al., 1978) do exist. Monoclonal antibodies have been prepared that are useful in the sandwich ELISA for the detection of SEB, SECs, SEA and SEE by one set of two antibodies for each group. Efforts to prepare monoclonal antibodies that cross-react with SEA, SED and SEE were generally unsuccessful, although an

Enterotoxigenicity of Staphylococcus aureus from Anterior Nares of Dining Hall Workers

The frequency of isolation of enterotoxigenic Staphylococcus aureus in dining hall workers of a Nigerian University was determined. Of a total of 186 workers sampled, 47 (25.3%) were carriers of enterotoxigenic S. aureus in their anterior nares, including 19 (22.4%) of 85 cooks and 11 (23.9%) of 46 stewards. Fifty-five (26.6%) of 207 strains of S. aureus tested produced staphylococcal enterotoxins A (SEA), B (SEB), C (SEC), D (SED) or E (SEE). SEA predominated, with 18 (8.7%) strains elaborating it and representing 32.7% of all enterotoxigenic strains. SEC and SED were produced by 14 (6.8%) and 13 (6.3%) strains, respectively, and 9 (4.3%) strains produced SEB and SEE. It appears that SEA poses the greatest risk to students consuming foods contaminated by S. aureus of nasal origin from these workers.

Enterotoxigenicity of Staphylococcus aureus Strains Isolated from Nigerian Ready-to-Eat Foods

The incidence of enterotoxin production by Staphylococcus aureus strains isolated from five Nigerian ready-to-eat products, i.e., roasted beef, dried beef, dried fish, fried fish and fried chicken, was investigated. Ninety-seven (39.1%) strains of 248 tested elaborated staphylococcal enterotoxins A(SEA), B(SEB), C(SEC), D(SED) or E(SEE). SED was most frequently produced as 43(17.3%) strains produced it, representing 44.3% of all enterotoxigenic strains. SEA and SEE were elaborated by 31(12.5%) and 29(11.7%) strains, respectively. Nineteen (7.7%) isolates produced SEB and the incidence of SEC was lowest, with only 12(4.8%) strains positive. Six (50.0%) of the SEC-producers were dried beef isolates. Of the three food sources (beef, fish and chicken) investigated, there was no significant difference (P>0.05, X2) between the incidence of enteroxigenicity of isolates, although beef isolates demonstrated the highest incidence (43.3%) followed by fish products (37.0%) and chicken product (36.0%). Regardless of source, roast beef isolates were the most frequently toxic (55.2%), whereas dried fish isolates were least frequently enterotoxigenic (34.5%). Multiple enterotoxin production most frequently encountered were types ADE, AD and BD which were produced by 6, 5 and 3 strains, respectively. It was concluded that the five ready-to-eat products investigated may pose a health hazard from the view point of staphylococcal intoxication due to the high incidence of enterotoxin production by isolates. SED appears to pose the greatest risk of this hazard to consumers of these products in this environment.