Effect of environmental stresses on antibody-based detection of Escherichia coli O157:H7, Salmonella enterica serotype Enteritidis and Listeria monocytogenes

AIMS: To study the reaction patterns of selected antibodies to Escherichia coli O157:H7, Salmonella enterica serotype Enteritidis and Listeria monocytogenes cells exposed to various environmental stresses. METHODS AND RESULTS: Escherichia coli O157:H7, Salmonella Enteritidis and L. monocytogenes cells subjected to different environmental stress of temperatures (4 and 45 degrees C), NaCl (5.5%), oxidative stress (15 mmol(-1) H2O2), acidic pH (5.5) and ethanol (5%) for 3 h (short-term stress) or for 5 days (long-term stress) were analysed by ELISA and Western blotting. The ELISA results indicated that most stresses caused 12-16% reductions in reaction for anti-E. coli O157:H7 and 20-48% reductions for anti-Salmonella polyclonal antibodies during short-term stress, whereas the most stresses exhibited enhanced reaction (44-100% increase) with the anti-L. monocytogenes polyclonal antibody. During long-term stress exposure to combined stress conditions of pH 5.5, 3.5% NaCl at 12 degrees C or at 4 degrees C, antibody reactions to the three pathogens were highly variable with the combined stress at 4 degrees C showing the most reductions (8-40%). Likewise, there were about 18-59% reductions in antibody reactions with pathogens when cultured in hotdog samples with the combined stress conditions. Western blot analyses of crude cell surface antigens from both short- and long-term stressed cells revealed that the changes in antibody reactions observed in ELISA were either because of repression, expression or possible denaturation of antigens on the surface of cells. CONCLUSIONS: Overall, the antibody reactions were significantly reduced in pathogens exposed to both short- and long-term environmental stresses in culture medium or in meat sample because of expression, repression or denaturation of specific antigens in cells. SIGNIFICANCE AND IMPACT OF THE STUDY: In order to ensure the reliable detection of foodborne pathogens using antibody-based methods, the influence of stress on antibody reactions should be thoroughly examined and understood first as the physiological activities in cells are often altered in response to a stress.     

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).     

Development of a piezoelectric immunosensor for the detection of Salmonella typhimurium.

A piezoelectric biosensor has been developed for the detection of Salmonella typhimurium. The antibody to Salmonella was immobilized on the crystal by various immobilization procedures. The best result was obtained when antibody was immobilized on the crystal precoated with a thin layer of polyethyleneimine. The response of the coated crystal for S. typhimurium in a microbial suspension was in the range of 10(5) to 10(9) cells ml-1. The time required for a complete interaction between the crystal and the cells appeared to depend upon the cell concentration of the analyzed sample. The antibody-bound crystal lost no activity over 4 days at 4 degrees C and it could be reused for 6-8 consecutive assays.     

Carcinoscorpius rotunda cauda amoebocyte lysate for detection of endotoxins--its preparation, stability, sensitivity and comparison with Limulus amoebocyte lysate

Carcinoscorpius amoebocyte lysate (CAL) was prepared from C. rotunda cauda by a modification of the method described by Mahalanabis et al. [Indian J Med Res, 70 (1979) 35]. Seasonal variation as well as batch variation was observed in the yield of haemolymph and the total lysate protein. In the presence of E. coli lipopolysaccharide (pure, free endotoxin) and E. coli and Salmonella cell suspensions (bound endotoxin), the CAL formed a gel after incubation at 37 degrees C. The gelling time varied from 10-90 min depending on the concentration of endotoxin used; higher concentrations formed gel more rapidly. The endotoxin detection capacity (sensitivity) of the lysate preparations was influenced by the season in which prepared, but not by the total protein content. Ten fold increase in the sensitivity was achieved by a purification step using chloroform. Although subsequent frozen storage with or without lyophilization did not alter the initial sensitivity, it was either decreased considerably or lost totally when the lysate was stored for 4 months at 4 degrees C or for 2 months at 30 degrees C. Under the same conditions, Limulus lysate was more stable. The lost sensitivity could not be regained by the incorporation of divalent cations (Ca2+ and Mg2+). The CAL preparations in general were able to detect as little as 10-100 pg of endotoxin or as few as 10(3) cells of E. coli or 10(4) cells of Salmonella and were comparable to LAL. CAL could be used successfully in lieu of Limulus amoebocyte lysate in the detection and assay of endotoxins.     

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.     

Automated detection of Salmonella spp. in foods.

An automated method to detect salmonellae in foods was developed and tested in food samples intentionally contaminated with the test organisms. Liquid eggs, shell eggs, dry eggs, skim milk and chicken were spiked with Salmonella enteritidis, S. typhimurium or S. newport to yield 2 to 25 CFU per 25 g or ml of sample. Following pre-enrichment in universal pre-enrichment broth at 42 degrees C for 6 h (eggs and milk) or 16 h (chicken), Salmonella cells were captured by immunomagnetic beads coated with Salmonella antibody (Vicam, Watertown, MA). The beads were transferred to selective liquid media containing carbohydrate (dulcitol or xylose), amino acid (lysine or ornithine), and H2S indicator, and incubated at 42 degrees C in the BioSys instrument (MicroSys, Ann Arbor, MI). Salmonella positive samples were identified by black discoloration of the media during incubation, while negative samples remained colorless. These color changes were recorded by the instrument. All the artificially contaminated samples tested positive within 15-18 h, while control samples remained negative during 24 h incubation. The results agreed with standard identification procedures. A total of 24 h was required to detect 2 to 25 CFU of the pathogen in 25 g or ml of eggs and milk, and up to 36 h in chicken, compared to 72 h in the standard methods.     

Monoclonal antibody storage conditions, and concentration effects on immunohistochemical specificity

Monoclonal antibodies against a 24,000 dalton intracellular estrogen-regulated protein in human breast cancer cells were used to study storage conditions and the effects of monoclonal antibody concentrations on immunohistochemical antigen localization. Both hybridoma supernatants and ascites fluid obtained from mice injected with hybridoma cells were used as sources of monoclonal antibodies; the monoclonal antibodies in the ascites fluid were concentrated and purified. Both antibody preparations were stored at 4, -20, or -70 degrees C and periodically tested for activity at these storage conditions. There was no difference in activity for the antibodies between storage at -20 and -70 degrees C. However, when highly diluted antibody was stored at 4 degrees C, the activity was lost within 2 weeks if carrier proteins were not added. These monoclonal antibodies were applied to immunohistochemical staining of different mouse and human tissues processed for routine paraffin sections, using the avidin-biotin-peroxidase procedure. A monoclonal antibody of unrelated specificity was used as control. When these antibodies were used at high concentrations, all the different tissues examined were immunostained. With reduction of the antibody concentration, an immunohistochemical dissection of the tissues was seen until specific immunostaining was reached. When even more highly diluted monoclonal antibody was used, heterogeneity in the staining pattern became very high. On the basis of these results, certain immunohistochemical criteria are proposed for the selection of the optimum concentration of monoclonal antibodies for specific antigen detection.     

Monoclonal antibodies to Salmonella lipopolysaccharide: anti-O-polysaccharide antibodies protect C3H mice against challenge with virulent Salmonella typhimurium

The present investigation reports the production of monoclonal antibodies to antigenic determinants of the O-polysaccharide of Salmonella typhimurium lipopolysaccharide (LPS), and assesses the effectiveness of these antibodies in protecting C3H mice against the lethal effects of Salmonella infection. Hybridomas were generated by fusing spleen cells from (BALB/c X A/J)F1 (CAF1) mice hyperimmunized by i.v. injection with acetone-killed S. typhimurium SR-11 with X63-Ag8.653 murine myeloma cells. Hybridomas producing antibodies reactive with S. typhimurium SR-11 whole cells were subcloned, and seven of the resulting clones as well as one previously described clone were selected for use in the studies reported here. Monoclonal antibodies from these eight clones were of the IgG1 (1), IgG3 (6), or IgM (1) isotype and were specific for the O-polysaccharide region of Salmonella LPS, reacting with LPS from smooth S. typhimurium SR-11 and LT-2, but not with LPS from rough S. minnesota R60 (Ra), R345 (Rb), or R595 (Re). The effectiveness of each monoclonal antibody in protecting C3H/HeN and C3H/HeJ mice against the lethal effects of Salmonella infection was evaluated by comparing the median length of survival of groups of mice given antibody by i.p. injection before i.p. challenge with virulent S. typhimurium SR-11 to that of animals that received no antibody. Three out of eight monoclonal anti-O-polysaccharide antibodies, ST-1 (IgM), 10-5-47 (IgG3), and 10-5-6 (IgG3), provided significant (p less than 0.01) protection to C3H/HeN mice challenged with approximately 10(4) LD100 of Salmonella. Only antibodies ST-1 and 10-5-6, however, extended the median length of survival of C3H/HeJ mice beyond that of infected controls. Mouse antiserum prepared against S. typhimurium SR-11 was equally protective in C3H/HeJ mice. In an attempt to understand the contribution of antibody specificity to the relative differences in the protective capacities of the monoclonal antibodies, their reactivities with several Salmonella reference strains of different classical serotypes were examined. Although some differences in reactivity against the different strains were apparent, this approach was not adequate for defining the fine specificity of these monoclonal antibodies. The results of this study provide evidence that monoclonal antibodies with specificity to the O-polysaccharide region of Salmonella LPS can protect C3H mice against challenge with the homologous bacterial strain.     

Quantification of mRNA in Salmonella sp. seeded soil and chicken manure using magnetic capture hybridization RT-PCR

Direct quantification of mRNA from Salmonella sp. seeded for 1 h to soil and chicken manure was accomplished using magnetic capture hybridization as a purification technique. This detection strategy targeted the invA gene present in Salmonella sp. After cell lysis, phenol/chloroform purification and isopropanol precipitation, the RNA extract was combined with the hybridization probe conjugated to paramagnetic beads. After hybridization, the captured nucleic acids were released by denaturation and purified of contaminating DNA using DNase. The resulting RNA was of high purity and there was no need for dilution of the samples prior to RT-PCR. The developed procedure was reproducibly used to quantify Salmonella sp. in high organic agricultural soil. The detection limit for mRNA using ordinary quantitative PCR (employing SYBRgreen-based detection) was 5 x 10(4)Salmonella sp. cells per gram of soil. Chicken manure amended into soil (1:4 w/w) did not reduce the ability to quantify Salmonella sp. mRNA in soil. Pasteurization (65 degrees C, 30 min) of chicken manure containing Salmonella sp. dramatically reduced the detection of invA mRNA (requiring 42 qPCR cycles for detection versus 26 cycles in unpasteurized manure), presumably due to degradation of the invA mRNA in Salmonella sp. cells killed by pasteurization. By contrast, DNA-based qPCR still detected Salmonella sp. in the pasteurized manure. Thus, in this case using samples seeded with fresh Salmonella sp. the mRNA-based detection appears to be superior to minimizing false-positive detection which was prevalent with DNA-based qPCR.     

Immunocytochemistry of cell surface heparan sulfate proteoglycan in mouse tissues. A light and electron microscopic study

The core protein of the proteoglycan at the cell surface of NMuMG mouse mammary epithelial cells bears both heparan and chondroitin sulfate chains and is recognized by the monoclonal antibody 281-2. Using this antibody and the peroxidase-antiperoxidase staining technique in adult mouse tissues, we found that the antibody recognizes the antigen in a highly restricted distribution, staining a variety of epithelial cells but no cells derived from embryonic mesoderm or neural crest. The antibody fails to stain any stromal (mesenchymal) or neuronal cells, with the exception of plasma cells and Leydig cells. Squamous and transitional epithelia stain intensely over their entire surfaces, whereas cuboidal and columnar epithelia stain moderately and only at the lateral surface of the basal cells. Within squamous and transitional epithelial tissues that undergo physiological regeneration (e.g., epidermis), the most superficial and differentiated cell types fail to stain. Within glandular and branched epithelia (e.g., pancreas), the secretory alveolar cells fail to stain. When evaluated by electron microscopy, granular deposits of stain are seen on the plasma membrane, especially on lateral surfaces, but none are noted within the cells or the basement membrane. These results indicate that in adult tissues the core protein of this heparan sulfate-rich proteoglycan is expressed almost exclusively at epithelial cell surfaces. Expression appears to be lost as the cells become either mature or highly differentiated.     

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.     

Developmental changes in unique cell surface antigens of chick embryo spinal motoneurons and ganglion cells

The monoclonal antibody technique was used to investigate neuronal heterogeneity and its developmental changes in the chick embryo trunk especially at the thoracic level. We report here four monoclonal antibodies (called SC 1, SC 2, SC 3, and SC 4) that bound to cell surface antigens. These antigens appeared to be proteins or glycoproteins because of their susceptibility to trypsin. In the spinal cord, antibody SC 3 stained all cells, but antibody SC 1 specifically stained motoneurons and ventral epithelial cells. The staining of motoneurons by antibody SC 1 was transient. It appeared at early stages (stage 16-17; Hamburger and Hamilton), but decreased markedly in intensity at older stages (stage 30-31). Antibody SC 2 did not stain cells in the spinal cord. It stained only neurons in the dorsal root and sympathetic ganglia. Antibody SC 4 stained only cells derived from the neural crest at the early stages (stage 16-20). At later stages, it stained a wider population of cells, including sensory neurons, Schwann cells, and cells in the central nervous system. In the dorsal root ganglion, antibodies SC 1 and SC 2 stained only neuronal cells whereas antibodies SC 3 and SC 4 stained both neuronal and glial cells. The dorsal root ganglionic antigens recognized by these antibodies were not expressed concurrently but appeared in a developmental sequence. Staining with antibodies SC 3 and SC 4 appeared first, then SC 1, and finally SC 2. Among these four antigens, the antigens common to both neuronal and glial cells appeared earlier than the neuron specific antigens. Thus, our monoclonal antibodies revealed heterogeneities in cell surface neuronal molecules and their transient and sequential appearance during embryonic development.     

Development and optimization of a novel immunomagnetic separation- bacteriophage assay for detection of Salmonella enterica serovar enteritidis in broth

Salmonella is the second-leading cause of food-borne illness in most developed countries, causing diarrhea, cramps, vomiting, and often fever. Many rapid methods are available for detection of Salmonella in foods, but these methods are often insensitive or expensive or require a high degree of technical ability to perform. In this paper we describe development and characterization of a novel assay that utilizes the normal infection cycle of bacteriophage SJ2 for detection of Salmonella enterica serovar Enteritidis in broth. The assay consists of four main stages: (i) capture and concentration of target cells by using immunomagnetic separation (IMS); (ii) infection of the target bacterium with phage; (iii) amplification and recovery of progeny phage; and (iv) assay of progeny phage on the basis of their effect on a healthy population of host cells (signal-amplifying cells). The end point of the assay can be determined by using either fluorescence or optical density measurements. The detection limit of the assay in broth is less than 10(4) CFU/ml, and the assay can be performed in 4 to 5 h. The results of this study demonstrate that the IMS-bacteriophage assay is a rapid, simple, and sensitive technique for detection of Salmonella serovar Enteritidis in broth cultures which can be applied to preenriched food samples.     

Effect of ovary holding temperature and time on equine granulosa cell apoptosis, oocyte chromatin configuration and cumulus morphology

The effects of ovary holding time and temperature on granulosa cell apoptosis, oocyte chromatin configuration and cumulus morphology were investigated through a series of experiments. Three experiments were performed to determine the effect of ovary holding time and temperature on granulosa cell apoptosis. Ovaries were held (1) at 20, 30 or 35-37 degrees C for up to 2h, (2) at 30 degrees C for 0-1, 1-2, 2-3, 3-4, 4-6 or 6-10h, and (3) granulosa cells were held for 0, 1, 2, 3, 5, 12 or 24h in M199 with Hank's salts at room temperature (suboptimal incubation). Granulosa cell DNA was analysed by ethidium bromide staining or 3'-end labelling. Two experiments were performed to determine the effect of ovary holding time and temperature on oocyte chromatin configuration. Ovaries were held (1) at 20, 30 or 35-37 degrees C for up to 3h and (2) at 20-37 degrees C for 0-1, 1-2, 2-3, 3-4, 4-6, 6-8 or 8-12h. The oocytes were stained with Hoechst stain 33258 and the chromatin configuration was evaluated. Two experiments were performed to determine the effect of ovary holding time and temperature on cumulus oophorus morphology. Ovaries were held at (1) 20-30 or 35-37 degrees C for up to 2h and (2) for 0-2, 2-4, 4-6, and 6-10h at 35-37 degrees C. The cumulus oocyte complex (COC) were retrieved and the cumulus morphology was evaluated. There was no difference in proportion of follicles with non-apoptotic granulosa cells in the two groups below body temperature (20 and 30 degrees C), but more follicles had apoptotic granulosa cells when the ovaries were held at 35-37 degrees C (P < 0.001). Holding ovaries at 30 degrees C for more than 3h increased the proportion of follicles with apoptotic granulosa cells (P < 0.01). When follicles with non-apoptotic granulosa cells were incubated at room temperature, there was no granulosa cell apoptosis in any of the follicles within the first 3h, but at 5h apoptosis was present in the granulosa cells of 22% of the follicles, and 78% of the follicles contained apoptotic granulosa cells at 24h (P < 0.001). The temperature at which the ovaries were held did not influence oocyte chromatin, although there was a tendency towards more condensed chromatin configurations in the groups below body temperature. More denuded and expanded COCs were present in the lower temperature group (P < 0.001). Oocyte chromatin configuration changed after 6h of holding (P < 0.001), and numbers of compact COCs decreased after 2h (P < 0.05). The present studies suggest that equine follicles should be held for no more than 3h at 20-30 degrees C if granulosa cell apoptosis is to be avoided. To avoid changes in cumulus oophorus morphology, ovaries should be held at 35-37 degrees C and for less than 2h before processing, and to avoid oocyte chromatin configuration changes, ovaries should be stored for less than 6h. When ovaries are to be used in oocyte maturation studies, and assuming that (1) CC is the chromatin configuration of choice for oocyte maturation, (2) that presence of granulosa cell apoptosis promotes maturation of the oocyte and (3) that expanded cumulus oocytes are preferable, the present data suggests that ovaries should be stored for 4-6h before oocyte retrieval.     

Cryopreservation, actin localization and thermotropic phase transitions in ram spermatozoa

The effects of controlled stress, i.e. cooling, upon the distribution of actin in ram spermatozoa were examined to investigate the hypothesis that cytoskeletal proteins are involved in the maintenance of sperm plasma membrane integrity. The normal distribution of actin on the spermatozoon was initially determined. A monoclonal antibody (IgM) interacted exclusively with the post-acrosomal region and the principal piece of the flagellum. By the use of a polyclonal antibody, actin was detected on the acrosome (excluding the equatorial segment), the post-acrosomal region and the whole of the flagellum. The actin was present in its non-filamentous form. Spermatozoa fixed at 39 degrees C and then treated for the immunofluorescent detection of actin with the monoclonal antibody were mostly unstained (proportion stained = 4.4% (+/- 1.6; s.e.m.); n = 8 ejaculates). Provided spermatozoa were permeabilized by greater than 0.025% Triton X-100 before immunofluorescence, actin was localized in the postacrosomal region of all sperm heads, and to a minor extent on the principal piece of the flagellum. Use of the polyclonal antibody confirmed that the post-acrosomal antigen was unmasked by detergent treatment. Slow cooling, over 2-h periods to various temperatures between 5 and 15 degrees C, also induced an increase in the proportion of cells showing post-acrosomal actin immunoreactivity. Cooling through the temperature range 15 to 10 degrees C markedly increased the proportion of immunoreactive cells (mean +/- s.e.m.; 12 +/- 4.5% at 15 degrees C; 27 +/- 4.5% at 10 degrees C; n = 4 ejaculates). Further cooling to 5 degrees C failed to elicit increased staining. Ultrastructural examination of cooled spermatozoa confirmed that a subpopulation of spermatozoa exhibited post-acrosomal actin immunoreactivity after cooling. These results are compatible with the suggestion that actin fulfills a stabilizing function in spermatozoa.     

Improved immunological membrane filter method for detection of food-borne Salmonella strains.

An improved membrane filter method that involves the use of an enzyme-labeled antibody stain has been developed for the rapid detection of Salmonella species in foods. The procedure is carried out directly on a hydrophobic grid-membrane filter without requiring transfer by blotting to nitrocellulose. Pure cultures of 54 Salmonella species and 10 foods artificially contaminated with Salmonella colindale gave a positive reaction in which Salmonella colonies were visible as purple dots. Of 11 nonsalmonella organisms, only Citrobacter freundii reacted with Spicer-Edwards antiserum. Of 22 naturally contaminated food samples, 10 were positive for both the hydrophobic grid-membrane filter procedure of the Association of Official Analytical Chemists and the improved enzyme-labeled antibody stain method, and there was perfect agreement between the methods. Of these 10 positive samples, one was negative by the Health Protection Branch method; of the negative samples, two were positive by this latter method. The improved enzyme-labeled antibody stain method allows detection of Salmonella spp. in foods within 48 h, requires little equipment, and is inexpensive, easy to perform, and suitable for automated detection.     

Improved immunological membrane filter method for detection of food-borne Salmonella strains

An improved membrane filter method that involves the use of an enzyme-labeled antibody stain has been developed for the rapid detection of Salmonella species in foods. The procedure is carried out directly on a hydrophobic grid-membrane filter without requiring transfer by blotting to nitrocellulose. Pure cultures of 54 Salmonella species and 10 foods artificially contaminated with Salmonella colindale gave a positive reaction in which Salmonella colonies were visible as purple dots. Of 11 nonsalmonella organisms, only Citrobacter freundii reacted with Spicer-Edwards antiserum. Of 22 naturally contaminated food samples, 10 were positive for both the hydrophobic grid-membrane filter procedure of the Association of Official Analytical Chemists and the improved enzyme-labeled antibody stain method, and there was perfect agreement between the methods. Of these 10 positive samples, one was negative by the Health Protection Branch method; of the negative samples, two were positive by this latter method. The improved enzyme-labeled antibody stain method allows detection of Salmonella spp. in foods within 48 h, requires little equipment, and is inexpensive, easy to perform, and suitable for automated detection.     

Immunosensor for the differentiation and detection of Salmonella species based on a quartz crystal microbalance

Immunosensors based on the microgravimetric quartz crystal microbalance (QCM) technique have been developed for the detection of Salmonella species from serogroups A, B and D. Salmonella serogroup-specific murine monoclonal antibodies, respectively, raised against these serogroups were immobilized onto the silver electrodes of piezoelectric (PZ) crystals by cross-linkage via glutaraldehyde (GA) to the electrode surfaces pre-coated with thin polyethyleneimine (PEI) layer. The specific immunosensors developed gave responses in linear ranges from 10(5) to 5x10(8) cells per ml with no significant interference from other strains of Salmonella and Escherichia coli up to 10(8) cells per ml. They showed good repeatability and excellent linear range, achieving detection limits down to 10(4) cells per ml with ability to distinguish different strains of Salmonella. These biosensors exhibited an exquisite specificity evidenced by their ability to discriminate antigens, the structures of which differ only by the isomeric form of di-deoxyhexose. The antibody-modified crystals showed no loss in activity over 4 days under storage at 4 degrees C.     

Evaluation of sperm acrosome reaction in the Asiatic elephant

This study focuses on the effect of chemicals on acrosome reaction in elephant spermatozoa. Semen was collected at the Washington Park Zoo in Portland, Oregon, from an 11-yr-old Asian elephant by artificial vagina (7 ejaculates) and transported to Mahidol University in Bangkok in extender at 4 to 5 degrees C within 24 to 28 h. A total of 500 x 10(6) sperm/mL was used for the control and for each of the 4 treatment groups: 1) cAMP (0.1 mM); 2) caffeine (0.1 mM); 3) Penicillamine hypotaurine and epinephine, PHE (penicillamine 2 mM, hypotaurine 1 mM, epinephine 1 mM); and 4) heparin (10 microg/mL) at 39 degrees C for 2 h. Aliquots were removed and the sperm viability, abnormal morphology, and acrosome status were evaluated by triple stain technique. Transmission electron microscopy (TEM) was used to observe changes of the sperm head membrane in all treatment groups. Trypan blue reliably stained dead spermatozoa, while rose Bengal stained only the spermatozoa with intact acrosomes. The concentration of dead sperm cells was similar in the 4 groups. The percentages of live acrosome-reacted spermatozoa in the control and in groups treated with caffeine, PHE, cAMP and heparin were 19.5 +/- 4.3, 38.1 +/- 4.0, 34.8 +/- 3.7, 29.8 +/- 0.8 and 28.0 +/- 4.2, respectively. The acrosome reaction rate was higher in the treatment groups than in the control (P<0.05). Caffeine and PHE caused significantly higher acrosome reaction of the sperm head than cAMP or heparin (P<0.05). The electron micrographs showed that the acrosome reaction occurred by the presence of apical vesiculation. The results indicated that 1) the triple stain technique allowed for evaluation of both viability and acrosome reaction simultaneously in elephant spermatozoa,2) acrosome reaction occurred at a high rate in all 3 treatment groups. 3) the effects of caffeine and PHE were significantly higher (P<0.05) than of cAMP and heparin, and 4) the data obtained from the triple stain technique corresponded to those from TEM.     

Rapid detection of salmonellae in poultry with the magnetic immuno-polymerase chain reaction assay.

Rapid detection of salmonellae in chicken meat was accomplished by using the magnetic immuno-polymerase chain reaction assay (MIPA). A direct polymerase chain reaction assay performed with chicken meat spiked with Salmonella typhimurium resulted in poor sensitivity (approximately 10(7) CFU/g of meat). The use of immunoseparation with a Salmonella serogroup B-specific monoclonal antibody improved the sensitivity, but enrichment was required for the detection of low levels of contamination. Enrichment for 6 h in either buffered peptone water, lactose broth containing tergitol-7, or selenite-cystine broth resulted in the detection of an initial inoculum of 100 CFU per g of meat. Enrichment of the salmonellae present on 25 g of spiked chicken meat for 24 h in either buffered peptone water or selenite-cystine broth before detection by the MIPA yielded a detection limit of approximately 0.1 CFU/g of meat. A detection limit of approximately 1 CFU/g of meat was obtained when the spiked meat was stored at -20 degrees C before enrichment for 24 h and analysis with the MIPA. Although the MIPA was developed for S. typhimurium, a MIPA in which a panel of six monoclonal antibodies specific for Salmonella serogroups A through E was used detected the presence of 0.1 CFU of Salmonella enteritidis per g of chicken meat. These data indicate that the method is applicable to other commonly isolated serotypes.