Phage restriction and the presence of small plasmids in Salmonella enteritidis

Sequence variation within the variable region of the 16S rRNA at position 440 to 480 allowed the synthesis of specific PCR primers for the identification of groups within the species Photorhabdus luminescens, symbionts of entomopathogenic nematodes of the genus Heterorhabditis. For the second PCR primer the highly conserved region at 755 to 795 was used. The P. luminescens type strain specific primer could not recognize any other P. luminescens strain. The primer TEMPERATUS based on the sequence of strain DSM12190 (isolated from North West European H. megidis strain HSH2) identified all P. luminescens associated with H. megidis from North West Europe and two isolates from closely the related nematode strains from Ireland. The primer TROPICUS based on strain DSM12191 (isolated from the nematode type strain H. indica strain LN2) identified P. luminescens of tropical origin isolated from H. indica. Symbionts of H. bacteriophora could not yet be separated into well described groups with the primers used. A comparison of sequence data resulted in the identification of additional groups. The non-symbiotic P. luminescens isolates are distinct in the variable region. The group HELIOTHIDIS contains 15 P. luminescens associated with H. bacteriophora from North East America. The MARELATUS group contains symbionts of the nematode H. marelatus from the West Coast of the US. The data together with the specific symbiotic association of P. luminescens strains with different nematode species support the division of the taxon P. luminescens into different species.     

Salmonella enteritidis colonization in turkey poults

An experimental trial on the colonization of two Salmonella enteritidis strains (phage types 4 and 8) originating from meat turkey flocks, were carried out. Three-day old poults were inoculated orally with approximately 10(6) cfu/bird (one group with PT8 and the other with PT4). clinical signs were not observed in any of the groups. The total reisolation rates from cloacal swabs in birds inoculated with PT4 was higher than from birds inoculated with PT8. The inoculated strains could also be detected in contact birds. Examination of internal organs 21 days p.i. revealed higher isolation rates in the group infected with PT4 than with PT8. The results indicate that both phage types of S. enteritidis were able to colonize the intestinal tract and the internal organs of turkey poults. However, the colonization and duration of shedding differed between the two phage types. This may be due to the fact that PT8 strain used was free from the S. e. virulence plasmid 37 MDa.     

Interaction of dexamethasone and Salmonella enteritidis immune lymphokines on Salmonella enteritidis organ invasion and in vitro polymorphonuclear leukocyte function

OBJECTIVE: To evaluate the effects of gemeprost on utero-placental and luteal circulation and on the embryo/fetus in normal first trimester pregnancies. STUDY DESIGN: Sixty-seven women with a normal first trimester pregnancy requesting termination of pregnancy for psychosocial reasons were randomly allocated to pre-operative treatment with vaginal suppositories containing placebo or gemeprost. The women underwent transvaginal color and spectral Doppler ultrasound examination before the application of the suppository, 4 h after the application of the suppository but before the abortion, and on the seventh post-operative day. Blood flow velocities in the uterine and subchorionic arteries, the intrachorionic area and arteries in the wall of the corpus luteum and the embryonic/fetal heart rate were measured. RESULTS: The median value for pulsatility index (PI) in the dominant uterine artery was 2.4 before treatment with gemeprost and 8.5 4 h after treatment (P = 0.0006); the corresponding values for time-averaged maximum velocity (TAMXV) being 27 cm/s and 10 cm/s (P = 0.0006). Four (14%) of 28 embryos/fetuses in the gemeprost group were dead 4 h after treatment with gemeprost and the median heart rate of those still alive was significantly lower than before treatment (130 vs. 163 bpm; P = 0.003). In the placebo group, the results for the uterine arteries and the embryonic/fetal heart rate did not differ significantly between the first and second ultrasound examinations. The median values for PI and TAMXV in the arteries of the corpus luteum wall at the first ultrasound examination were 0.71 and 18 cm/s, respectively, in the placebo group and 0.71 and 20 cm/s, respectively, in the gemeprost group. These values remained almost unchanged at the second and third ultrasound examinations in both groups. CONCLUSION: Gemeprost has profound effects on utero-placental circulation in the first trimester and can induce embryonic/fetal bradycardia and sometimes embryonic/fetal demise. It has no unequivocal effect on luteal circulation.     

Phage typing combined with pulsed-field gel electrophoresis and random amplified polymorphic DNA increases discrimination in the epidemiological analysis of Salmonella enteritidis strains

In adult rabbits, mid-diaphyseal segments of the radius or ulna were excised to produce defects greater than the critical size for spontaneous bone repair. The defects were enveloped in sleeves composed of nonbiodegradable expanded polyfluoroethylene (ePTFE), pore size 30, 60, 90 microns, and compared with sleeves of three biodegradable materials. Bone morphogenetic protein and associated noncollagenous bone matrix protein (BMP/NCP) or recombinant human morphogenetic protein (rhBMP-2) were implanted inside the sleeves. Albumin was implanted for a control system. Without intracompartmental BMP, only about 10%-15% of the defect was repaired by bone growth extending from the bone ends into the sleeves composed of ePTFE, pore size 30 microns. With sleeves with pore size 60 or 90 microns and intracompartmental BMP/NCP, 54%-96% regeneration occurred within 8 weeks after the operation. Sleeves of biodegradable nonimmunogenic materials such as polyorthoester (POE) and polylactic-polyglycollic acids (PLA/PGA) permitted 86%-98% restoration of bone continuity, but only when BMP was present in the lumen. With puncture holes (0.5 mm in diameter), implants of BMP/NCP in the 30-micron PTFE sleeve produced transmembrane external callus formation and bone regeneration to 147%. Sleeves composed of aorta first calcified, then induced complete intracompartmental bone regeneration. Atelocollagen sleeves incited a low-grade inflammatory cell reaction and did not promote complete regeneration. Under conditions presently undisclosed segments of the ulna bridged with ePTFE, were incompletely paired, even with intracompartmental BMP/NCP. Puncture holes of 0.5 mm admitted ingrowth of capillaries and introduced local conditions favorable for the response to BMP/NCP. BMP/NCP may promote proliferation of nutrient vessels and differentiation of bone marrow stroma cells between the open bone ends. For further investigation, the hypothesis to be examined is that the optimum response to BMP/NCP and rhBMP-2 would emerge in compartments containing first a high concentration gradient and second proliferating perivascular cells.     

Periplasmic and fimbrial SefA from Salmonella enteritidis

Salmonella enteritidis produces thin, filamentous fimbriae composed of the fimbrin subunit SefA. Although insoluble in most detergents and chaotropic agents, these fimbriae were soluble at pH 10.5. Furthermore, in sodium dodecyl sulfate, these fibers depolymerized into monomers, dimers and other multimers of SefA, which precipitated on removal of the detergent. In contrast, unassembled periplasmic SefA fimbrins purified from Escherichia coli expressing cloned sefA and sefB were readily soluble in aqueous solution. Fimbrial and periplasmic SefA also differed in their reaction with an anti-SEF14 monoclonal antibody and in their surface hydrophobicity, indicating that the two forms had different properties. Precise mass measurements of periplasmic and fimbrial SefA by mass spectroscopy showed that these variations were not due to post-translational modifications. Periplasmic SefA consisted primarily of intact as well as some N-terminally truncated forms. The main 24 amino acid, N-terminally truncated form of periplasmic SefA was present as a 12.2 kDa monomer which had a low tendency to dimerize whereas intact periplasmic SefA was present as a 34.1 kDa homodimer. Intact periplasmic SefA also formed stable multimers at low concentrations of chemical cross-linker but multimerization of the truncated form required high concentrations of protein or cross-linker. Thus, SefA fimbrins appear to multimerize through their N-termini and undergo a conformational change prior to assembly into fibers. Within these fibers, subunit-subunit contact is maintained through strong hydrophobic interactions.     

Genotypic characterization of Salmonella enteritidis phage types by plasmid analysis, ribotyping, and pulsed-field gel electrophoresis

Pulsed-field gel electrophoresis (PFGE) was used to resolve XbaI and SpeI macrorestriction fragments from 60 defined phage type (PT) reference strains of Salmonella enteritidis. The level of discrimination was compared to that afforded by plasmid profile analysis and ribotyping. Twenty-eight distinct XbaI pulsed-field profiles (PFPs) were observed, although a single type, PFP X1, predominated. Absence of the 57-kb spv-associated fragment was observed for three PT reference strains, and the profile was designated PFP X1A. The XbaI macrorestriction profiles of a further four PT reference strains were altered by the presence of plasmid-associated bands. Twenty-six SpeI-generated PFPs (plus one subtype) were observed for the same strains. No SpeI fragment corresponding to the 38-MDa serovar-specific plasmid was detected. The distribution of XbaI and SpeI profiles did not always correspond, producing a total of 32 combined PFPs for the 60 PT reference strains. This compared with a total of 18 different plasmid profiles and three PvuII ribotypes generated by the same strains. The results of this study indicate that PFGE may offer an improved level of discrimination over other genotypic typing methods for the epidemiological typing of S. enteritidis.     

Evaluation of bacterins containing three predominant phage types of Salmonella enteritidis for prevention of infection in egg-laying chickens

Six Salmonella enteritidis bacterin formulations differing in adjuvant content and whole-cell inactivation procedures were evaluated in egg-laying chickens. Chickens given S enteritidis bacterins containing modified Freund's incomplete adjuvant had greater humoral immune responses to S enteritidis than did birds given other bacterin formulations (P < 0.05). Better protection against infection by S enteritidis phage types 8, 13a, and 23 was obtained in birds vaccinated with bacterin 5. Bacterin 5 contained S enteritidis cells inactivated by 20% acetone and modified Freund's incomplete adjuvant.     

Detection of Salmonella enteritidis in suspected flocks of laying hens

In the present paper, we report that an inositolphosphoglycan (IPG), derived from a Trypanosoma cruzi glycoinositolphosphoceramide (LPPG), is able to inhibit ACTH-mediated accumulation of a glucocorticoid, cortisol, in calf adrenocortical cells. This IPG is also able to inhibit the stimulation by ACTH of the production of the main glucocorticoid, corticosterone and the main mineralocorticoid, aldosterone, in rat adrenocortical cells. Nitrous acid deamination confirmed that IPG is responsible for this inhibition. In order to study the involvement of glycosylphosphatidylinositol (GPI) in ACTH response in rat adrenal cortex, the activation of a phospholipase that hydrolyzes GPI (GPI-PLC) was evaluated. It was found that the release of alkaline phosphatase, a GPI-anchored enzyme, to the extracellular medium is increased in rat adrenocortical cells by ACTH treatment. In addition, ACTH stimulates the release of ceramide from the glycoinositolphosphoceramide purified from T. cruzi. These data suggest that ACTH activates a GPI-PLC in rat adrenal cortex, which is in agreement with our previous data in calf adrenocortical cells; thus, the hydrolysis of GPI provoked by ACTH takes place in different mammals and the IPG released could inhibit ACTH-mediated synthesis of aldosterone, corticosterone and cortisol.     

Efficacy of a live avirulent Salmonella typhimurium vaccine in preventing colonization and invasion of laying hens by Salmonella typhimurium and Salmonella enteritidis

An avirulent live delta cya delta crp Salmonella typhimurium strain chi 3985 that precludes colonization and invasion of chickens by homologous and heterologous Salmonella serotypes was evaluated for its long-term protection efficacy. Chickens vaccinated orally at 2 and 4 wk of age were assessed for protection against oral challenge with wild-type S. typhimurium and Salmonella enteritidis strains at 3, 6, 9, and 12 mo of age. A comparison of Salmonella isolation from vaccinated and nonvaccinated layers after challenge with S. typhimurium or S. enteritidis showed that delta cya delta crp S. typhimurium chi 3985 induced excellent protection against intestinal, visceral, reproductive tract, and egg colonization, invasion, and/or contamination by Salmonella. The duration of protection lasted for 11 mo after vaccination, at which time the experiment was terminated. S. enteritidis and S. typhimurium were isolated from the yolk, albumen, and shells of eggs laid by nonvaccinated chickens challenged with Salmonella. S. typhimurium caused pathological lesions in nonvaccinated chickens, whereas vaccinated and nonvaccinated chickens challenged with S. enteritidis showed no pathological lesion in the visceral and reproductive organs. Vaccination with chi 3985 prevented transmission of S. typhimurium or S. enteritidis into eggs laid by vaccinated layers with no effect on egg production. To our knowledge, this is the first publication confirming that vaccination with live avirulent Salmonella can induce long-term protection against Salmonella infection in layers.     

Detection of Salmonella enteritidis in eggs by the polymerase chain reaction

A polymerase chain reaction (PCR) for the specific detection of the gene sequence, sefA, encoded by all isolates of Salmonella enteritidis, was developed. The PCR could detect as few as four S enteritidis washed bacterial cells but egg contents inhibited the PCR. Eggs spiked with 50 S enteritidis bacterial cells were homogenised, inoculated into buffered peptone water and grown at 37 degrees C for 16 hours, when the PCR was successful. A positive internal control was developed to differentiate between true and false negative PCR results for the detection of S enteritidis. In a limited trial of the egg handling procedures and the PCR, one of 250 chickens' eggs from retail outlets was found to be contaminated with S enteritidis.     

Molecular analysis of Salmonella enteritidis by pulsed-field gel electrophoresis and ribotyping

A total of 61 isolates of Salmonella enteritidis were analyzed by the techniques of pulsed-field gel electrophoresis (PFGE) and ribotyping. Twenty-three of the isolates were from Zurich, Switzerland, and 38 isolates were from the University Hospital, Kuala Lumpur, Malaysia. Five of the Malaysian isolates were hospital-related outbreak strains and were shown to be indistinguishable by PFGE analysis following digestion with three different restriction endonucleases, XbaI (5'-TCTAGA-3'), SpeI (5'-ACTAGT-3'), and AvrII (5'-CCTAGG-3'). The PFGE pattern of an isolate from a suspected carrier staff nurse was found to be identical to those of the hospital outbreak isolates. These isolates were also indistinguishable by ribotyping with SmaI and SphI. The same single PFGE pattern was also detected in 29 of 32 sporadic isolates of S. enteritidis. Four closely related ribotypes were detected among these 29 isolates. Similarly, outbreak-related strains from Switzerland showed close genetic identity by PFGE and ribotyping. Strains obtained from poultry showed more variations in their PFGE patterns and ribotypes, although the patterns were still closely related. In addition, SphI ribotypes A and D among the Swiss strains correlated with phage types 4 and 8, respectively. No correlation of phage types with PFGE pattern was noted. Both PFGE and ribotyping indicate that the S. enteritidis strains circulating in Malaysia and Switzerland are very similar and may be clonally related. Comparison of the PFGE patterns with the ribotypes for 23 Swiss and 16 Malaysian isolates showed that there was a 69% concordance in the grouping of isolates. We conclude that the close genetic similarity observed between epidemiologically unrelated and outbreak-related isolates of S. enteritidis suggests that both PFGE and ribotyping are of limited value in the epidemiological analysis of these particular isolates, possibly because of the highly clonal nature of pathogenic strains of S. enteritidis.     

Epidemiology and control of egg-associated Salmonella enteritidis in the United States of America

The isolation rate for Salmonella enterica serotype Enteritidis (SE) in humans in the United States of America (USA) increased from 1,207 sporadic isolates identified in 1976 (0.6 isolates/100,000 population) to 10,201 identified in 1995 (4.0/100,000 population). The proportion of reported Salmonella isolates which were SE increased from 5% to 25% during the same time period. In 1990, 1994, and 1995, SE was the most commonly reported reported Salmonella serotype in the USA. Much of this increase has been associated with the consumption of contaminated shell eggs. An examination of the results of a United States Department of Agriculture (USDA) survey of spent hens at slaughter and unpasteurised liquid egg at breaker plants (liquid egg processors) in 1991 and 1995 reveals an increase in the prevalence of SE isolates overall and in most regions of the USA. SE phage type 4 (pt 4), the predominant SE phage type in other parts of the world, has emerged in the egg industry in the western USA concurrent with a sharp increase in the number of sporadic human SE pt 4 isolates in California and Utah. Research on the molecular structure and virulence of SE pt 4 isolates from the USA as compared with isolates from other parts of the world (human and poultry) should be a priority. A comparison of DNA from pt 4 isolates from the USA and Europe may provide information about the potential threat to public health and poultry in the USA from this phage type. Some regional success in the reduction of human illness as a result of SE control efforts is apparent. The Pennsylvania Egg Quality Assurance Program has shown progress in reducing SE infection in participating flocks. At a national level, however, neither the incidence of human illness due to SE nor the prevalence of SE in flocks and unpasteurised liquid eggs have decreased significantly, despite the implementation of the USDA 'trace back' regulation from 1990 to 1995, and intensified efforts to educate food handlers and to enforce safe food handling practices. More effort is needed to control SE at every stage of the egg continuum, from production through to consumption. A risk-reduction approach, with barriers to the introduction and multiplication of the pathogen throughout the farm-to-table continuum, is the most practical method for reducing human illness from SE in shell eggs at present. An effective long-term solution will require interdisciplinary efforts involving government, industry, consumers, and academics. Interventions should be developed and evaluated in compliance with the potential for reducing the risk to human health and cost-effectiveness.     

Analysis of splenic and thymic lymphocyte subpopulations in chickens infected with Salmonella enteritidis

Lymphocytes expressing CD3, CD4, CD8, pan lymphocyte, IgA, IgG and IgM cell surface antigens were assessed by in the spleen and thymus of chickens following infection with Salmonella enteritidis using flow cytometric analysis. At 6 days post primary infection and 2 days post secondary infection with S. enteritidis, the percentages of IgA+ and IgM+ lymphocytes in the spleen were significantly increased (P < 0.05). At 2 days post secondary infection with S. enteritidis, the percentage of CD4+ T lymphocyte in the spleen and CD8+ T lymphocyte percentage in the thymus were significantly increased (P < 0.05). These results indicate that S. enteritidis infection induces changes in the spleen and thymus that reflect the dynamics of the host protective immune response.     

Supraclavicular abscess caused by Salmonella enteritidis in patient without gastrointestinal symptoms

Soft tissue infections are among the rare manifestations of extra intestinal salmonellosis and occur more frequently in immunocompromised patients. Herein we report a case of a 51-year-old white male with type II. diabetes mellitus, diagnosed with supraclavicular abscess caused by Salmonella enteritidis. The patient denied any gastrointestinal symptoms, and stool cultures were negative. After incision, drainage, and administration of intravenous ampicillin (4 x 1.5 g/day for two weeks) the patient recovered completely. To our knowledge, this is the first reported case of cervical soft tissue abscess caused by Salmonella enteritidis without preceding gastrointestinal symptoms. We feel that the presence of diabetes was a risk factor for developing the infection.     

An outbreak of Salmonella enteritidis in a maternity and neonatal intensive care unit

We investigated the relationships between methylphenidate (MPD) enantiomers and endogenous dopamine (DA) levels in striatal extracellular fluid, and that between DA level and locomotor activity, after intravenous administration of racemic MPD (2, 5 or 10 mg/kg dose) or the individual enantiomers (2.5 mg/kg dose) to rats. MPD and DA levels in the extracellular fluid were measured by in vivo brain microdialysis. The maximum levels of MPD enantiomers in the striatal extracellular fluid were obtained within 15 min after administration. On the other hand, the mean maximum DA levels after administration of 2-10 mg/kg dose of racemic MPD were obtained within 10 min with values in the range of 3.0- to 8.6-fold higher than the basal DA level. The maximum DA level (4.2-fold of the basal level) after administration of (+/-)-MPD was greater than that (2.2-fold) of the same dose of (-)-MPD. A clockwise hysteresis was observed between MPD concentration and DA level in the extracellular fluid after MPD administration. Locomotor activity after administration of (+)-MPD was also greater than (-)-MPD. From these results, it was shown that the locomotor activity induced by MPD may be related to the increase of DA level in the extracellular fluid, and the degree of increase of the DA level by (+)-MPD was greater than that of the (-)-isomer.     

Chilling invokes different morphologies in two Salmonella enteritidis PT4 strains

The reduction in chemical preservatives in food processing has resulted in more refrigerated (chilled) products. However, the effect of chilling on Salmonella enteritidis PT4 isolates has received relatively little attention. This study investigates the effect of chilling on two Salm. enteritidis PT4 isolates, denoted E and I. These isolates differ in their tolerance to heat, acidification, survival on surfaces, and behaviour in animal models. E routinely shows greater tolerance and pathogenicity than I. Chilling invokes profound cell elongation and heterogeneity in E which corresponded to a 90% sublethal injury; neither such substantial cell elongation nor significant injury was seen in I. The ability to recover resistance to desoxycholate coincided with a reduction to normal cell size. Incomplete cell division and failure of the septum to form is a likely hypothesis for cell elongation although outer membrane changes could be responsible. Possible links are suggested between cell elongation of the hat- and acid-tolerant strain and pathogenicity.     

Maternal and neonatal lead exposure in southern Italy

BACKGROUND: Over the past 20 years, there has been remarkable improvement in the chances of survival of patients treated in burn centers. A simple, accurate system for objectively estimating the probability of death would be useful in counseling patients and making medical decisions. METHODS: We conducted a retrospective review of all 1665 patients with acute burn injuries admitted from 1990 to 1994 to Massachusetts General Hospital and the Shriners Burns Institute in Boston. Using logistic-regression analysis, we developed probability estimates for the prediction of mortality based on a minimal set of well-defined variables. The resulting mortality formula was used to determine whether changes in mortality have occurred since 1984, and it was tested prospectively on all 530 patients with acute burn injuries admitted in 1995 or 1996. RESULTS: Of the 1665 patients (mean [+/-SD] age, 21+/-20 years; mean burn size, 14+/-20 percent of body-surface area), 1598 (96 percent) lived to discharge. The mean length of stay was 21+/-29 days. Three risk factors for death were identified: age greater than 60 years, more than 40 percent of body-surface area burned, and inhalation injury. The mortality formula we developed predicts 0.3 percent, 3 percent, 33 percent, or approximately 90 percent mortality, depending on whether zero, one, two, or three risk factors are present. The results of the prospective test of the formula were similar. A large increase in the proportion of patients who chose not to be resuscitated complicated comparisons of mortality over time. CONCLUSIONS: The probability of mortality after burns is low and can be predicted soon after injury on the basis of simple, objective clinical criteria.