In 2009, four bathing sites in The Netherlands were monitored for potentially human pathogenic
Vibrio species to observe possible associations with environmental conditions and health complaints. ...Three slightly different enrichment procedures were used to isolate
Vibrio species with different growth requirements. Waters were generally positive for
Vibrio from May until October; median
Vibrio concentrations ranged from 4 to 383 MPN per litre (maximum 10
5 MPN per litre). Isolated
Vibrio species included
V. alginolyticus (50.6%) and
V. parahaemolyticus (8.5%) from bathing sites with salinities ranging between 2.8 and 3.5% and
V. cholerae non-O1/O139 (6.9%) from sites with salinities ranging between 0.007 and 0.08%. Although more samples were positive for
Vibrio at elevated water temperatures, a quantitative relation between
Vibrio numbers in water samples and the water temperature was not observed which may be explained by maximum water temperatures of 21
°C. Active surveillance yielded one case of a recreational water related
Vibrio infection.
V. cholerae non-O1/O139 was cultured from the patient's wound and the implicated recreational water; PFGE profiles of the water and patient isolates were not identical. The number of patients that contract a
Vibrio infection through exposure to Dutch recreational waters seems low, but may be underestimated. The common occurrence of
Vibrio species in these waters stresses the need for providing information on
Vibrio to risk groups to prevent infections.
ABSTRACT
A high‐throughput biosafety level 3 suitable method is necessary for rapid discovery of unknown pathogens in complex matrices from the food chain. In this paper, three automated bacterial ...DNA extraction methods for real‐time polymerase chain reaction (PCR) detection were evaluated. The systems compared were the Magnatrix (NorDiag, Oslo, Norway) and the EZ1 (Qiagen, Hilden, Germany), both based on purification with magnetic beads, and the ABI Prism™ 6100 Nucleic Acid PrepStation (Applied Biosystems, Foster City, CA), which is based on silica gel filtration. In the comparison, the detection of bacterial DNA from food and feed seeded with one Gram‐positive and one Gram‐negative bacterial species was compared. A wider range of matrices were successfully processed using the magnetic bead‐based DNA extraction robots compared with the silica gel filtration‐based robot. The limit of detection was 102–103 cfu/g matrix of both bacteria in powdered milk, minced meat, whole wheat and soypass using the EZ1 extraction robot.
PRACTICAL APPLICATIONS
A rapid, wide‐range method to discover unknown dangerous pathogens in complex matrices from the food chain is crucial to minimize the effects in the society of extraordinary events. These events are defined as accidents or unexpected disease outbreaks and deliberate spread of highly pathogenic bacteria. Gaining knowledge of the advantages and disadvantages of extraction methods from complex matrices is important in the development of fast and reliable detection methods.
Experimental oral infection of pigs with a parental Yersinia pseudotuberculosis strain pIB102, serotype O:3 and two mutant isogenic strains - pIB155,ΔyopK and pIB44,ΔypkA has been carried out. ...Clinical findings, microbiological and immunological parameters were examined in dynamics from day 7 to day 60 post-infection (p.i.). All types of infections ran asymptomatically, without hyperthermia, loss of appetite, etc. Experiments on the blood parameters demonstrated a transient leucocytosis with lymphocytosis and monocytosis better expressed after yopK infection. Even though pig is usually known as a reservoir of yersiniae, bacterial colonization was found in mesenterial lymph nodes and tonsils on day 7, respectively 14 p.i. with parental strain, and only in tonsils on day 14 p.i. with both mutant strains. The augmented sensitivity of mutants to the bactericidal effect of leukocytes and blood sera is the characteristic feature of attenuation in their pathogenicity, compared to the parental strain. Comparative in vitro experiments on the immune response and immunostimulating capacity of Y. pseudotuberculosis mutant strains verify their preserved immunogenic potential, predominantly in case of yopK. Hyperplasia and strong activation of the lymph tissue of Peyer's patches, mesenterial lymph nodes, tonsils and spleen of pigs challenged with both mutant strains were proved as immunomorphological rearrangements. The results obtained give the reason to claim that the genetically constructed yopK null mutant strain is significantly attenuated but is still immunogenic and has the potential for a live vaccine carrier strain.
Yersinia enterocolitica employs a type III secretion system (TTSS) to target virulence factors (e.g. YopE) into the cytosol of the host cells. We utilized the TTSS to introduce a recombinant antigen ...directly into the cytosol of host cells and to investigate the potential of
Y. enterocolitica and
Y. pseudotuberculosis as live carrier for vaccines. The model antigen ovalbumin (Ova) was fused to defined secretion or translocation domains of the
Yersinia effector protein YopE and introduced into attenuated mutant strains of
Y. enterocolitica and
Y. pseudotuberculosis. In vitro experiments showed secretion and translocation of YopE-Ova hybrid proteins into host cells. To investigate the resulting immune responses, mice expressing transgenic Ova-specific T cell receptors were used. Both
Y. enterocolitica and
Y. pseudotuberculosis mutants induced efficaciously Ova-specific CD8
+ T cell responses. The translocation domain of YopE was required for induction of CD8
+ T cell responses in vivo, but not for T cell responses induced in vitro. The in vivo frequency of Ova-specific splenic T cells was up to six-fold higher in mice immunized with YopE-Ova-translocating
Y. enterocolitica/Y. pseudotuberculosis mutants than in control mice. The Ova-specific T cells were shown to produce high amounts of IFN-gamma. We did not observe significant Ova-specific CD4
+ T cell or antibody responses upon vaccination with either of the strains. In conclusion,
Yersinia live carrier vaccine strains are suitable to target antigens into the MHC class I pathway and stimulate CD8
+ T cell responses and thus, might be useful in vaccine approaches against intracellular pathogens.
Experimental oral infections of rabbits with a wild-type Yersinia pseudotuberculosis strain (pIB102), and two null-mutants (yopK and ypkA) were carried out with the aim to explore the possibility to ...use mutant strains of Y. pseudotuberculosis as live carrier vaccine strains. The infectious process of the three strains proceed with passing hyperthermia, leucocytosis with granulocytosis, moderate monocytosis and a transient lymphopenia, better demonstrated at mutant strain infections. Short-term bacterial dissemination into the brain and viscera was observed at yopK infection. An augmented resistance to bactericidal activity of leucocytes at the initial phase of infection was followed by an increased sensitivity discovered earlier in case of yopK strain accompanied by at least 70- and 20-fold, respectively, for ypkA lower virulence for mice. The level of attenuation of yopK was accompanied by significant Yersinia specific IgG and IgM antibody response. Inflammatory foci were found by morphological examination in brain, lung and small intestines after infection with the wild-type strain, while such foci were only observed in brain and mesenterial lymph nodes after infection with the yopK mutant. After infection with the ypkA mutant foci were found in brain and spleen of the infected animals. Morphological changes in the lymphatic tissue of rabbits infected with mutant strains were consistent with induction of immunogenesis. The data suggest that genetically constructed yopK null-mutant exhibits characteristics that makes the strain suitable to be used as a live carrier vaccine to deliver heterologous antigens.
The cellular and molecular mechanisms that underlie species-specific membrane fusion between male and female gametes remain largely unknown. Here, by use of gene discovery methods in the green alga ...Chlamydomonas, gene disruption in the rodent malaria parasite Plasmodium berghei, and distinctive features of fertilization in both organisms, we report discovery of a mechanism that accounts for a conserved protein required for gamete fusion. A screen for fusion mutants in Chlamydomonas identified a homolog of HAP2, an Arabidopsis sterility gene. Moreover, HAP2 disruption in Plasmodium blocked fertilization and thereby mosquito transmission of malaria. HAP2 localizes at the fusion site of Chlamydomonas minus gametes, yet Chlamydomonas minus and Plasmodium hap2 male gametes retain the ability, using other, species-limited proteins, to form tight prefusion membrane attachments with their respective gamete partners. Membrane dye experiments show that HAP2 is essential for membrane merger. Thus, in two distantly related eukaryotes, species-limited proteins govern access to a conserved protein essential for membrane fusion.
Extracellular Yersinia pseudotuberculosis employs a type III secretion system (T3SS) for translocating virulence factors (Yersinia outer proteins Yops) directly into the cytosol of eukaryotic cells. ...Recently, we used YopE as a carrier molecule for T3SS-dependent secretion and translocation of listeriolysin O (LLO) from Listeria monocytogenes. We demonstrated that translocation of chimeric YopE/LLO into the cytosol of macrophages by Yersinia results in the induction of a codominant antigen-specific CD4 and CD8 T-cell response in orally immunized mice. In this study, we addressed the requirements for processing and major histocompatibility complex (MHC) class II presentation of chimeric YopE proteins translocated into the cytosol of macrophages by the Yersinia T3SS. Our data demonstrate the ability of Yersinia to counteract exogenous MHC class II antigen presentation of secreted hybrid YopE by the action of wild-type YopE and YopH. In the absence of exogenous MHC class II antigen presentation, an alternative pathway was identified for YopE fusion proteins originating in the cytosol. This endogenous antigen-processing pathway was sensitive to inhibitors of phagolysosomal acidification and macroautophagy, but it did not require the function either of the proteasome or of transporters associated with antigen processing. Thus, by an autophagy-dependent mechanism, macrophages are able to compensate for the YopE/YopH-mediated inhibition of the endosomal MHC class II antigen presentation pathway for exogenous antigens. This is the first report demonstrating that autophagy might enable the host to mount an MHC class II-restricted CD4 T-cell response against translocated bacterial virulence factors. We provide critical new insights into the interaction between the mammalian immune system and a human pathogen.
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