12 research outputs found
Intestinal–mucosal IgA anti–LPS and serum vibriocidal antibody responses elicited by two rounds of oral immunizations in Balb/c mice two weeks apart with formalin–killed MS1568 and MS1580 whole cell vaccines as compared to Dukoral vaccines; immunizations and collection of tissue specimens are fully described in Materials and Methods.
<p>(A) IgA anti–LPS antibody levels in fecal extracts (expressed as units per mg of total IgA measured by ELISA); and (B) the same in small intestinal tissue extracts. (C) Serum vibriocidal antibody responses against Inaba test organisms; and (D) the same against Ogawa test organisms. Bars represent the pooled results (geometric mean values ± SEM) from two separate experiments in Balb/c mice, each with 8 animals per group. Analyses of data by ANOVA showed that post–immunization antibody levels did not differ significantly between any of the immunization groups.</p
Determinations of the relative amounts of Ogawa antigen in LPS preparations of the generated Hikojima strains MS1568 and MS1580 based on amounts of methylated and un–methylated perosamine analyzed with mass spectrometry in relation to similar analyses of LPS preparations from reference Inaba Phil6973 and Ogawa Cairo 50 strains.
<p>The area under the curve for the methylated part (m/z ratio 756.6) divided by the area under the curve for the non–methylated part (m/z ratio 742.6) subtracted with the background (this ratio from the Inaba strain Phil6973) was used for the two Hikojima strain (MS1568 and MS1580) LPS preparations to calculate their percentage of Ogawa LPS (compared to the 100% Ogawa in the LPS from the Cairo50 reference strain). Diagrams are from one of 3 such experiments showing closely similar patterns and with the calculated mean ± SEM percentages Ogawa antigen indicated for MS1568 and MS1580.</p
Colony blot results.
<p>O1 Inaba <i>V. cholerae</i> strain JS1569 was transformed with expression plasmids carrying different mutant <i>wbeT</i> genes. Different mutations in the <i>wbeT</i> gene give different levels of expression of the Ogawa antigen as seen by different levels of staining following labelling with Ogawa–specific antibodies. The plasmids were isolated and the <i>wbeT</i> genes sequenced. The mutations present in the different clones are indicated.</p
Comparison of intestinal–mucosal IgA anti–LPS and serum vibriocidal antibody responses elicited by oral immunization with formalin–killed MS1568 or Dukoral vaccines in CD1 mice.
<p>(A) IgA anti–LPS antibody levels measured by ELISA in fecal extracts (dashed) and in small intestinal tissue extracts (striped) after two rounds of intragastric immunizations and (B) same after three rounds as described in Material and Methods. (C) Serum vibriocidal antibody responses against Inaba (filled) and Ogawa (open) test organisms after two and (D) three rounds of immunizations. Bars show geometric mean values and SEM for 7 animals per group. As tested with ANOVA, post–immunization antibody levels did not differ significantly between any of the different immunization groups.</p
A schematic figure over the construction of the suicide plasmid and its incorporation into and it’s the removal from the genome.
<p>(A) Amplification of the mutated <i>wbeT</i> gene the upstream DNA was generated to be fused together in a primerless PCR. (B) The fused sequenced were used in another primerless PCR together with the kanamycin resistant gene flaked by FRT–sites and the downstream sequenced of <i>wbeT</i> gene. (C) The result of the second primerless PCR used to be incorporated into the pMT–suicide–sacB plasmid for integration into the genome of O1 <i>V. cholerae</i>. (D) Primer orientation of the wbe region before and (E) after gene manipulation.</p
Primer and Oligonucleotide used for cloning and analysis of the wbeT gene.
<p>Primer and Oligonucleotide used for cloning and analysis of the wbeT gene.</p
Bacterial strains and plasmids used in the current study.
<p>Bacterial strains and plasmids used in the current study.</p
Cholera cases recorded in municipal wards 29, 30 and 33 in Kolkata.
<p>All cases of cholera showing serotype of isolated strains, Ogawa (blue solid line) and Inaba (red dotted lined), over the period from March 2003 until the end of 2007, shown per month. The blue dashed line shows the cases recorded over an extended period within the same area of Kolkata until the end of 2011 [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005044#pntd.0005044.ref018" target="_blank">18</a>]. There are generally seasonal outbreaks with one in the spring and one in the autumn.</p
Schematic diagram of the <i>wbeT</i> gene showing mutations giving rise to the Inaba serotype in the current study.
<p>The insertions and the M1I mutations were confined to Clade III and the S158P mutation was confined to clade IV. In all there were 219 isolates in Clade III. The insertion at amino acid position 49 occurred in seven isolates and the insertions at 97 and 147 were present in a single isolate. Two strains were isolated with the M1I mutation. The remaining 209 isolates were Ogawa. In Clade IV there were 139 isolates. Of these 128 were Inaba due to the S158P mutation and 11 were Ogawa due to a reversion event. 5 out of the 128 Inaba isolates in Clade IV carried multiple insertions in addition to the S158P mutation (not shown).</p
The distribution of cholera cases.
<p>The three wards of Kolkata (29, 30 and 33) in which the study was conducted showing all the documented cases of cholera according to the GPS coordinates of the patients. Green X represents project health clinics, blue and red dots represent Ogawa and Inaba serotyped cases respectively.</p