Detection of Yersinia enterocolitica serogroup O:3 by a PCR method

Yersinia enterocolitica is the etiologic agent of a range of clinical situations in humans, but only a small number of serotypes are involved. Among these, Y. enterocolitica O:3 is the most frequently implicated. A PCR method was developed to detect Y. enterocolitica O:3. For this purpose, two pairs of primers were designed to amplify two fragments of the rfb cluster of Y. enterocolitica O:3: a 253-bp fragment of the rfbB gene and a 405-bp fragment of the rfbC gene. A specific detection was obtained only with rfbC primers, which yielded a PCR product of the expected size exclusively with pathogenic Y. enterocolitica of serotype O:3. This pair of primers was combined with the ail, inv, and virF primers previously described (H. Nakajima, M. Inoue, T. Mori, K.-I. Itoh, E. Arakawa, and H. Watanabe, J. Clin. Microbiol. 30:2484-2486, 1992) to allow both the detection and the differentiation between Y. pseudotuberculosis, pathogenic Y. enterocolitica of serotype O:3 and other pathogenic Y. enterocolitica.     

Comparison of siderophore production and utilization in pathogenic and environmental isolates of Yersinia enterocolitica

Yersinia enterocolitica strains of serotypes lethal to mice have been reported previously to produce an endogenous siderophore. In this study, an ethyl acetate-extractable siderophore was characterized and given the name yersiniophore. Yersiniophore was produced by 16 of 16 human isolates of serotypes O:4, O:4,32, O:8, O:21, and one nonhuman isolate of serotype O:21. It was not produced by isolates of serotype O:3, O:5, or O:9. One strain of Yersinia pseudotuberculosis produced yersiniophore, but strains of Yersinia kristensenii, Yersinia frederiksenii, and Yersinia intermedia did not produce or utilize yersiniophore. Food and water isolates of Y. enterocolitica produced a water-soluble siderophore but not yersiniophore. Sixty-two strains of Y. enterocolitica including 42 isolates from human infections, 2 animal isolates, and 18 water and food isolates were examined for utilization of yersiniophore, the water-soluble siderophore, and ferrioxamine. Yersiniophore promoted growth rate, iron binding, and uptake in 17 of 62 strains, all of which produced yersiniophore. Ten of 17 food and water isolates and one human isolate were capable of utilizing the water-soluble siderophore. Utilization studies suggest that at least one additional water-soluble siderophore may be produced. Ferrioxamine promoted the growth of 60 of 62 strains examined; however, only the 17 strains which produced yersiniophore actively accumulated [59Fe]ferrioxamine. Yersiniophore production and utilization may be important in clinical infections since all human strains belonging to serotype O:8 produced yersinophore. The water-soluble siderophore was not detected in human isolates.     

Phospholipase A of Yersinia enterocolitica contributes to pathogenesis in a mouse model

Some isolates of Yersinia enterocolitica exhibit phospholipase activity, which has been linked to lecithin-dependent hemolysis (M. Tsubokura, K. Otsoki, I. Shimohira, and H. Yamamoto, Infect. Immun. 25:939-942, 1979). A gene encoding Y. enterocolitica phospholipase was identified, and analysis of the nucleotide sequence revealed two tandemly transcribed open reading frames. The first, yplA, has 74% identity and 85% similarity to the phospholipase A found in Serratia liquefaciens. Though the other, yplB, was less similar to the downstream accessory protein found in S. liquefaciens, the organization in both species is similar. Subsequently, a yplA-null Y. enterocolitica strain, YEDS10, was constructed and demonstrated to be phospholipase negative by plate and spectrophotometric assays. To ascertain whether the phospholipase has a role in pathogenesis, YEDS10 was tested in the mouse model. In experiments with perorally infected BALB/c mice, fewer YEDS10 organisms were recovered from the mesenteric lymph nodes and Peyer's patches (PP) than the parental strain at 3 or 5 days postinfection. Furthermore, bowel tissue and PP infected with YEDS10 appeared to be less inflamed than those infected with the parental strain. When extremely high doses of both the parental and YEDS10 strains were given, similar numbers of viable bacteria were recovered from the PP and mesenteric lymph nodes on day 3. However, the numbers of foci and the extent of inflammation and necrosis within them were noticeably less for YEDS10 compared to the parental strain. Together these findings suggest that Y. enterocolitica produces a phospholipase A which has a role in pathogenesis.     

Selective isolation from HeLa cell lines of Yersinia pseudotuberculosis, pathogenic Y. enterocolitica and enteroinvasive Escherichia coli

Using HeLa cell lines, we obtained an optimal and selective isolation of Yersinia pseudotuberculosis, pathogenic Y. enterocolitica and enteroinvasive Escherichia coli from samples such as pork, feces and river water heavily contaminated with other bacteria.     

A mRNA signal for the type III secretion of Yop proteins by Yersinia enterocolitica

Pathogenic Yersinia species have a specialized secretion system (type III) to target cytotoxic Yop proteins during infection. The signals of YopE and YopN sufficient for the secretion of translational reporter fusions were mapped to the first 15 codons. No common amino acid or peptide sequence could be identified among the secretion signals. Systematic mutagenesis of the secretion signal yielded mutants defective in Yop translation; however, no point mutants could be identified that specifically abolished secretion. Frameshift mutations that completely altered the peptide sequences of these signals also failed to prevent secretion. Thus, the signal that leads to the type III secretion of Yop proteins appears to be encoded in their messenger RNA rather than the peptide sequence.     

Construction and characterisation of a Yersinia enterocolitica O:8 ompR mutant

OBJECTIVE: To test whether a barrier of chemically cross-linked pure hyaluronic acid reduces postoperative adhesion formation. DESIGN: The material was evaluated in the murine uterine horn model using excision and electrocautery injuries and in animals who had amounts of material inserted into the abdomen to evaluate toxicity. SETTING: Academic medical center. SUBJECT(S): Mice. INTERVENTION(S): Insertion of the barrier between uterine horns and into the peritoneal cavity. MAIN OUTCOME MEASURE(S): Adhesion formation at 14 days; the histology of the peritoneum, liver, and spleen at 42 days; and the number, differential count, morphology, and flow cytometry of peritoneal leukocytes 3 days postoperatively. RESULT(S): Fewer adhesions were present when excision injuries were separated by the barrier (12 of 28 sites [43%] versus 23 of 26 control sites [88%]), whereas the number of adhesions was unchanged after electrocautery injuries (14 of 26 sites [54%] versus 17 of 26 control sites [65%]). The uterine horn sites covered by the barrier were histologically indistinguishable from controls. No adverse impact on the peritoneum and peritoneal fluid leukocyte population was observed with barrier insertion. CONCLUSION(S): The use of a barrier composed of a chemically cross-linked hyaluronic acid derivative (Incert, Anika Therapeutics, Inc., Woburn, MA) reduced postoperative adhesion formation in this model without any adverse impact on the peritoneum and peritoneal leukocyte population. This barrier material shows promise in preventing postoperative adhesions and deserves clinical evaluation.     

Rapid shell vial culture technique for detection of enteroviruses and adenoviruses in fecal specimens: comparison with conventional virus isolation method

Detection of enteroviruses and adenoviruses mainly in fecal specimens by rapid culture with inoculation onto cell monolayers in flat-bottom tubes by centrifugation and immunofluorescence staining with genus-specific monoclonal antibodies was compared with that by the conventional virus isolation procedure. For both conventional culture and shell vial culture human lung fibroblast cells and tertiary monkey kidney cells were used. For enterovirus detection, 979 clinical specimens (916 stool specimens, 56 cerebrospinal fluid specimens, and 7 nasopharyngeal swabs) were used. Conventional culture detected 74 enterovirus isolates. A cytopathic effect compatible with the presence of an enterovirus after 3 days of incubation occurred in 25 of the 74 (34%) specimens that eventually became positive. The detection rate for enteroviruses by rapid cell culture after 2 to 3 days of incubation was 42 of 74 (57%). The genus-specific enterovirus monoclonal antibody did not react with strains of echovirus types 22 and 23 or enterovirus type 71. Rapid cell culture for the detection of adenoviruses was performed with 567 clinical specimens (536 stool specimens, 25 cerebrospinal fluid specimens, and 6 miscellaneous specimens), in which 42 adenoviruses were found by conventional culture. Nine of the 42 (21%) adenovirus isolates were detected by conventional culture within 3 days after inoculation, whereas 21 (50%) were found by rapid cell culture within 2 to 3 days. Only two of the nine specimens found to be positive for the enteric adenovirus type 41 by conventional culture as well by a type-specific enzyme-linked immunosorbent assay (ELISA) tested positive by rapid cell culture. In conclusion, the rapid shell vial assay allows the early detection and identification of enteroviruses and adenoviruses in clinical specimens but is markedly less sensitive than the conventional isolation procedure according to the eventual results of the conventional isolation procedure. Conventional cell culture remains a prerequisite for serotyping of enteroviral isolates. On the basis of the results for adenovirus type 41, the rapid detection of adenoviruses was not considered to be useful for the detection of clinically relevant adenoviruses in fecal samples.     

Vasculitis and bacteraemia with Yersinia enterocolitica in late-onset systemic lupus erythematosus

We report a case of Yersinia enterocolitica 0.9 septicaemia complicating systemic lupus erythematosus in an elderly male patient. The infection gave rise to digital vasculitis, fevers and general malaise on top of pre-existing articular symptoms. Features of Yersinia septicaemia may mimic some of the signs of lupus.     

Sources of sporadic Yersinia enterocolitica infections in Norway: a prospective case-control study

Yersinia enterocolitica is a recognized cause of gastroenteritis in northern Europe. During October 1988-January 1990, a prospective case-control study was performed to address risk factors associated with sporadic Y. enterocolitica infections in southeastern Norway. Sixty-seven case-patients (mean age 23.4 years, range 8 months-88 years) and 132 age-, sex- and geographically-matched controls were enrolled in the study. Multivariate analysis of the data showed that persons with Y. enterocolitica infection reported having eaten significantly more pork items (3.79 v. 2.30 meals, P = 0.02) and sausage (2.84 v. 2.20 meals, P = 0.03) in the 2 weeks before illness onset than their matched controls; only one patient had eaten raw pork. Patients were also more likely than controls to report a preference for eating meat prepared raw or rare (47 v. 27%, P = 0.01), and to report drinking untreated water (39 v. 25%, P = 0.01) in the 2 weeks before illness onset. Each of these factors was independently associated with disease, suggesting a link between yersiniosis and consumption of undercooked pork and sausage products and untreated water. Efforts should be directed towards developing techniques to reduce Y. enterocolitica contamination of pork and educating consumers about (1) proper handling and preparation of pork items and (2) the hazards of drinking untreated water.     

Yersinia enterocolitica infection in a 4-month-old infant associated with infection in household dogs

Colon atresia is a rare cause of intestinal obstruction in the neonate and requires early diagnosis and prompt surgical treatment. It is impossible in the neonate to differentiate colon atresia from other forms of obstruction at the time of initial presentation. The diagnosis is confirmed roentgenographically, including views of the abdomen and contrast barium enema series. Lesions proximal to the splenic flexure are treated with initial resection of the atretic segment and a primary anastomosis. Those lesions distal to the splentic flexure are managed initally with a diverting loop colostomy with subsequent staged resection and anastomosis.     

Shortened PCR cycles in a conventional thermal cycler

Although swallowing difficulties (dysphagia) frequently occur in acute brainstem infarction, physiological studies of dysphagia (videofluoroscopy, manometry) are rarely reported. We present a patient with ipsilateral Horner's syndrome, palatal and laryngeal weakness, aphagia, and ipsilateral face and contralateral extremity pin and temperature loss due to lateral medullary infarction confined to the rostral dorsolateral medulla (RDM). Videofluoroscopy showed that the patient was unable to initiate a swallow. Manometry showed a markedly reduced peak pharyngeal pressure and weak pharyngeal contractions. Within 20 months, the patient's neurological deficits resolved, videofluoroscopy showed a normal swallow, and manometry showed normal peak pharyngeal pressure. Correlation of the clinical, physiological, and imaging evaluations shows that aphagia and severe bilateral pharyngeal paresis can result from unilateral RDM infarction. We suggest that, in man, the bilateral medullary swallowing centers function as one integrated center, and that infarction of a portion of this center is sufficient to cause complete loss of swallowing.     

Experimental mixed infection with Yersinia enterocolitica and Listeria monocytogenes in guinea pigs

The pathogenesis and the cell immune response (CIR) of guinea pigs after mixed infection with Y. enterocolitica and L. monocytogenes was investigated. The guinea pigs were infected per os with 1.1 x 10(9) CFU Y. enterocolitica 0:3, (pYV+) and four days later with 1.1 x 10(9) CFU L. monocytogenes 4B. Clinical, paraclinical and morphological findings attending the infectious process were followed in dynamics up to the 28th day post infection (p.i.) with L. monocytogenes. The phagocyting activity of alveolar macrophages (aMa) was suppressed against Y. enterocolitica, in contrast to peritoneal macrophages (pMa) engulfing yersiniae more actively at the end of the study. Moreover, the tendency of augmented entering in both phagocytes of L. monocytogenes cells was well demonstrated, starting at the earlier intervals of examination. Histopathological studies showed a purulent meningoencephalitis and a catarrhal pneumonie, non-reactive micronecroses in the spleen and lymphadenitis catarrhalis in the mesenteric lymph nodes. Analysis of the T-cell immune response (T-CIR) showed maximal values in the spleen lymphocytes after Y. enterocolitica and L. monocytogenes mixed infection. The B-CIR occurred early (at the 7th day p.i.) and was maximal at the 28th day p.i. in blood lymphocytes. The results obtained demonstrated that the mixed infection of guinea pigs with Y. enterocolitica and L. monocytogenes runs has a non lethal, generalized illness with a dominant role of L. monocytogenes cells.     

The use of a surface adhesion immunofluorescent (SAIF) method for the rapid detection of Yersinia enterocolitica serotype O:3 in meat

Defective trafficking of the cystic fibrosis transmembrane conductance regulator (CFTR) is the most common cause of cystic fibrosis. In chloride-secreting epithelia, it is well established that CFTR localizes to intracellular organelles and to apical membranes. However, it is controversial whether secretagogues regulate the trafficking of CFTR. To investigate whether acute hormonal stimulation of chloride secretion is coupled to the trafficking of CFTR, we used the intact shark rectal gland, a model tissue in which salt secretion is dynamically regulated and both chloride secretion and cellular CFTR immunofluorescence can be quantified in parallel. In rectal glands perfused under basal conditions without secretagogues, Cl- secretion was 151+/-65 microeq/h/g. Vasoactive intestinal peptide (VIP), forskolin, and genistein led to 10-, 6-, and 4-fold increases in Cl- secretion. In basal glands, quantitative confocal microscopy revealed CFTR immunofluorescence extending from the apical membrane deeply into the cell (7.28+/-0.35 micron). During stimulation with secretagogues, apical extension of CFTR immunofluorescence into the cell was reduced significantly to 3.24+/-0.08 micron by VIP, 4.08+/-0.13 by forskolin, and 3.19+/-0.1 by genistein (P < 0.001). Moreover, the peak intensity of CFTR fluorescence shifted towards the apical membrane (peak fluorescence 2.5+/-0.13 micron basal vs. 1.51+/-0.06, 1.77+/-0.1, and 1.38+/-0.05 for VIP, forskolin, and genistein; all P < 0.001). The increase in both Cl- secretion and apical CFTR trafficking reversed to basal values after removal of VIP. These data provide the first quantitative morphological evidence for acute hormonal regulation of CFTR trafficking in an intact epithelial tissue.     

The tampon test for trichomoniasis: a comparison between conventional methods and a polymerase chain reaction for Trichomonas vaginalis in women

Vertebral artery tortuosity causing neural foraminal widening is a well described abnormality that should not be confused with other causes of neural foraminal enlargement, particularly on conventional roentgenograms. We, hereby, describe CT features of another cervical osseous change due to the vertebral artery tortuosity, the so called "tubular shaped vertebral artery canal", which is embedded in the vertebral body instead of causing neural foramen enlargement. Catheter and MR angiographic studies have also been performed to confirm the vertebral artery tortuosity causing the osseous changes.     

Novel DNA assay for cytomegalovirus detection: comparison with conventional culture and pp65 antigenemia assay

We compared conventional cytomegalovirus (CMV) isolation, rapid viral culture, a CMV pp65 antigenemia assay, and a novel CMV DNA hybrid capture system (HCS). A total of 309 blood samples from individuals in different risk groups were assessed by at least two of the methods mentioned above. Leukocytes were recovered either after centrifugation in Leucosep tubes containing 1.080 Ficoll for pp65 assay or after simple differential lysis steps for DNA detection. HCS was based on DNA hybridization with a CMV RNA probe and its capture by antibodies to DNA-RNA hybrids. The CMV pp65 lower matrix protein was detected by fluorescence with c10-c11 monoclonal antibody in formalin-fixed leukocytes. Concordant results were observed for 92.9, 78.3, and 82.7% of the patients when comparing (i) viral culture and the pp65 antigenemia assay, (ii) viral culture and HCS, and (iii) the pp65 antigenemia assay and HCS, respectively. Discordant results were observed between a positive HCS result and negative culture and/or pp65 results. These results were associated with relatively low DNA levels (< 20 pg/10(6) cells) and positive viruria. In conclusion, the pp65 antigenemia assay is a rapid and reliable method of detecting CMV and is preferable to culture, but the Murex HCS appears to be more sensitive for CMV detection.     

Irradiation as a method for decontaminating food. A review

In the present work we report the high-level expression of foreign genes encoding the light-harvesting (LHII) membrane-spanning polypeptides in photosynthetic bacteria. To do this we first constructed three deletion strains of Rhodovulum (Rhv.) sulfidophilum in which all or part of the puc operon, encoding the peripheral light-harvesting proteins, is missing. To investigate the heterologous expression of the light-harvesting polypeptides from Rb. capsulatus in Rhv. sulfidophilum and vice versa we have reintroduced functional foreign LH genes into these and equivalent strains of Rhodobacter (Rb.) capsulatus. In some cases very high levels of expression were obtained (85%) of those observed in the wild type), while in other cases much lower expression was observed; possible reasons for these differences are discussed. The heterologously expressed proteins were shown to contain normal pigment-binding sites and to be normally and functionally integrated within the host photosynthetic apparatus. The results indicate that heterologous proteins are able to assemble properly and enter into the same protein-protein interactions as their analogs originally present in the host strain.     

Immuno-PCR: a sensitive method for detecting antigen

A myotoxic phospholipase A2, bothropstoxin II, which exhibits low hydrolytic activity, was crystallized and X-ray diffraction data were collected to a resolution of 2.2 A. Preliminary analysis reveals the presence of three molecules in the asymmetric unit.