Recent and emerging innovations in Salmonella detection: a food and environmental perspective

Salmonella is a diverse genus of Gram‐negative bacilli and a major foodborne pathogen responsible for more than a million illnesses annually in the United States alone. Rapid, reliable detection and identification of this pathogen in food and environmental sources is key to safeguarding the food supply. Traditional microbiological culture techniques have been the ‘gold standard’ for State and Federal regulators. Unfortunately, the time to result is too long to effectively monitor foodstuffs, especially those with very short shelf lives. Advances in traditional microbiology and molecular biology over the past 25 years have greatly improved the speed at which this pathogen is detected. Nonetheless, food and environmental samples possess a distinctive set of challenges for these newer, more rapid methodologies. Furthermore, more detailed identification and subtyping strategies still rely heavily on the availability of a pure isolate. However, major shifts in DNA sequencing technologies are meeting this challenge by advancing the detection, identification and subtyping of Salmonella towards a culture‐independent diagnostic framework. This review will focus on current approaches and state‐of‐the‐art next‐generation advances in the detection, identification and subtyping of Salmonella from food and environmental sources.     

Impact of Marine Submergence and Season on Faunal Colonization and Decomposition of Pig Carcasses in the Salish Sea

Pig carcasses, as human proxies, were placed on the seabed at a depth of 300 m, in the Strait of Georgia and observed continuously by a remotely operated camera and instruments. Two carcasses were deployed in spring and two in fall utilizing Ocean Network Canada’s Victoria Experimental Network under the Sea (formerly VENUS) observatory. A trial experiment showed that bluntnose sixgill sharks could rapidly devour a carcass so a platform was designed which held two matched carcasses, one fully exposed, the other covered in a barred cage to protect it from sharks, while still allowing invertebrates and smaller vertebrates access. The carcasses were deployed under a frame which supported a video camera, and instruments which recorded oxygen, temperature, salinity, density, pressure, conductivity, sound speed and turbidity at per minute intervals. The spring exposed carcass was briefly fed upon by sharks, but they were inefficient feeders and lost interest after a few bites. Immediately after deployment, all carcasses, in both spring and fall, were very rapidly covered in vast numbers of lyssianassid amphipods. These skeletonized the carcasses by Day 3 in fall and Day 4 in spring. A dramatic, very localized drop in dissolved oxygen levels occurred in fall, exactly coinciding with the presence of the amphipods. Oxygen levels returned to normal once the amphipods dispersed. Either the physical presence of the amphipods or the sudden draw down of oxygen during their tenure, excluded other fauna. The amphipods fed from the inside out, removing the skin last. After the amphipods had receded, other fauna colonized such as spot shrimp and a few Dungeness crabs but by this time, all soft tissue had been removed. The amphipod activity caused major bioturbation in the local area and possible oxygen depletion. The spring deployment carcasses became covered in silt and a black film formed on them and on the silt above them whereas the fall bones remained uncovered and hence continued to be attractive to large numbers of spot shrimp. The carcass remains were recovered after 166 and 134 days respectively for further study.     

Characterization of an insulin-like growth factor binding protein, analogous to human pregnancy-associated secreted endometrial alpha 1-globulin, in decidua of the baboon (Papio anubis) placenta

The major secreted protein of the human decidua (pregnancy-associated endometrial alpha 1-globulin [alpha 1-PEG]), is an insulin-like growth factor-binding protein (IGF-BP) that is immunologically and biochemically similar to placental protein 12 (PP12) extracted from term human placenta. Since previous studies have demonstrated that the baboon and human endometrium synthesize and release a number of biochemically and immunologically related polypeptides in culture, this study was undertaken to further characterize a related IGF-BP in baboon placental tissues. Decidua, chorio-amniotic membranes with adhering decidua (CAM-D), and placental villi were obtained from pregnant baboons between Days 134 and 160 of gestation by Cesarean sections. Portions of tissue were either cultured in the presence of 35S-methionine, fixed for immunocytochemistry, or frozen in liquid nitrogen for cytosol extraction. Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) of tissue culture media (TCM) revealed that the major secretory product of the decidua and CAM-D was an acidic polypeptide (Mr 33,000). Western blot analysis and immunoprecipitation of TCM with murine monoclonal antibody (B2H10) against human alpha 1-PEG demonstrated that this molecule, secreted by the baboon decidua and CAM-D, but not the placental villi, was immunologically identical to the human IGF-BP. Immunocytochemical localization of IGF-BP was intense in the cytoplasm of stromal cells in decidua and CAM-D and absent in the placenta. Gel filtration of TCM and cytosol followed by screening of eluates for 125I-IGF-I binding resolved two peaks (Mr greater than 100,000 and 35,000) of specific IGF-BP in decidua and CAM-D. The 35,000 peak had 100-200 times the binding capacity of the Mr greater than 100,000 peak and a Kd of 1.14-1.83 nM. The eluates contained in the Mr 35,000 peak were also immunoreactive to alpha 1-PEG, as accessed by a polyclonal radioimmunoassay. Affinity cross-linking with 125I-IGF-I followed by sodium dodecyl sulfate-PAGE revealed an immunoreactive complex of Mr 36,000, confirming that the baboon protein represents a high affinity IGF-BP. These studies indicate that the hypertrophied stromal cells of the baboon decidua and CAM-D synthesize and release an IGF-BP as their major secretory product, analogous to the situation in humans. The results of this study suggest that this protein may play a role in the regulation of IGF action during pregnancy.     

Adenosine diphosphate-induced aggregation of human platelets in flow through tubes. II. Effect of shear rate, donor sex, and ADP concentration.

The effect of shear rate on the adenosine diphosphate-induced aggregation of human platelets in Poiseuille flow was studied using the method described in part I (Bell, D.N., S. Spain, and H.L. Goldsmith. 1989. Biophys. J. 56:817-828). The rate and extent of aggregation in citrated platelet-rich plasma were measured over a range of mean transit time from 0.2 to 8.6 s and mean tube shear rate, G, from 41.9 to 1,920 s-1. At 0.2 microM ADP, changes in the single platelet concentration with time suggest that more than one type of platelet-platelet bond mediates platelet aggregation at physiological shear rates. At low G, a high initial rate of aggregation reflects the formation of a weak bond of high affinity, the strength of which diminishes with time. Here, the fraction of collisions yielding stable doublets, the collision efficiency, reached a maximum of 26%. The collision efficiency decreased with increasing G and was accompanied by a progressive delay in the onset of aggregation. However, the gradual expression of a more shear rate-resistant bond at high shear rates and long mean transit times produced a subsequent increase in collision efficiency and a corresponding increase in the rate of aggregation. Although the collision efficiencies here were less than 1%, the high collision frequencies were able to sustain a high rate of aggregation. At 0.2 microM ADP, aggregate size generally decreased with increasing G. At 1.0 microM ADP, aggregate size was still limited at high shear rates even though the rate of single platelet aggregation was much higher than at 0.2 microM ADP. Platelet aggregation was greater for female than for male donors, an effect related to differences in the hematocrit of donors before preparing platelet-rich plasma.     

Localization of epidermal growth factor precursor in tooth and lung during embryonic mouse development

The murine epidermal growth factor (EGF) precursor is a 1217 amino acid protein which contains mature EGF (amino acid residues 977-1029) as well as eight EGF-like repeats. Although the highest levels of EGF are found in the adult male mouse submandibular gland, the results of in situ hybridization studies and mRNA analyses suggest that EGF precursor mRNA is synthesized in several adult mouse tissues including the lung and the incisor. To determine if EGF precursor gene expression is intrinsic to the developmental program for either embryonic tooth or lung organogenesis, sense and antisense oligodeoxyribonucleotide probes corresponding to amino acids 1070-1081 of the precursor were used to localize cellular sites of synthesis of EGF precursor mRNA by in situ hybridization. Antibodies directed against amino acid residues 348-691 of the precursor were used in immunodetection techniques to identify either EGF precursor protein or processed derivatives. In contrast to earlier reports indicating that embryonic mouse tissues do not synthesize EGF precursor mRNA, we found that EGF precursor mRNA is present in clusters of ectoderm-, mesoderm-, and ectomesenchyme-derived cells associated with embryonic teeth and lung organs. Moreover, epitopes common to the EGF precursor were immunolocalized in both the epithelial and mesenchymal tissues of embryonic mouse tooth and lung organs. These results suggest that the EGF precursor and/or motifs contained within the precursor molecule, including mature EGF, may play an instructive or permissive role in epithelial-mesenchymal interactions pursuant to organogenesis.     

Phosphorylation of Myelin-Associated Glycoprotein In Vivo and In Vitro Occurs Only in the Cytoplasmic Domain of the Large Isoform

Myelin-associated glycoprotein (MAG) was radioactively labelled with 32P both in intact brain and in myelin membrane preparations. Chemical deglycosylation of the phosphorylated products revealed that only one of the MAG isoforms (L-MAG) is labelled in vitro. Furthermore, the phosphorylation events in vivo and in vitro are confined to the cytoplasmic portion of the L-MAG isoform. Tryptic mapping of L-MAG labelled both in vivo and in vitro revealed that the majority of the sites phosphorylated in intact brain are also phosphorylated in myelin membrane preparations; however, the extent of phosphorylation at individual sites is variable. The results demonstrate that partially purified myelin membrane preparations can be used to study the enzymes responsible for MAG phosphorylation and dephosphorylation events in vivo.     

Brahman bull fertility in a north Australian rangeland herd

Low and variable bull fertility was identified as a constraint on reproductive rates in beef cattle grazed in an extensive, multiple sire mating regimen on Mount Bundey station in the Darwin pastoral district of northwestern Australia. Erratic conception patterns were attributed to a high proportion of bulls with low breeding soundness evaluation scores (BSE), a high proportion of aged bulls (40%>8 yr), and to running bulls of mixed age groups. Liveweight, scrotal circumference (SC) and age were positively correlated. An experiment was subsequently designed to investigate the ability of a number of bull measurements to predict fertility in an extensively-managed, multiple-sire mating system. Blood typing was used to match calves to sires. It proved to be an accurate and useful technique which successfully identified the parentage of 94% of calves examined. Single measurements of serum testosterone after administration of gonadotrophin-releasing hormone (GnRH) were not correlated with fertility. However six of the seven most fertile bulls exhibited high peak serum testosterone levels in summer, and lower levels in the winter. In contrast, the less fertile bulls did not exhibit seasonal variation in GnRH-induced serum testosterone levels. Social dominance ratio was weakly ralted to fertility (r=0.51: P<0.05). BSE (r=0.51: P<0.05) and SC (r=0.49: P<0.05) prior to, but not subsequent to, mating were correlated with bull fertility. Under the conditions of this experiment, a bull to cow ratio of 1:20 was excessive for bulls with a satisfactory BSE score.