B. anthracis alters its virulence gene expression profile in response to a number of environmental signals, including levels of bicarbonate and CO2. Virulence plasmid pXO1 is important to Bacillus anthracis pathogenicity as it carries the genes encoding the anthrax toxin and virulence regulatory factors. Induction of toxin and other virulence genes requires the pXO1-encoded AtxA regulatory protein. The cytochrome c maturation system influences the expression of virulence factors in Bacillus anthracis. B. anthracis carries two copies of the ccdA gene, encoding predicted thiol-disulfide oxidoreductases that contribute to cytochrome c maturation. Loss of both ccdA genes results in a reduction of cytochrome c production, an increase in virulence factor expression, and a reduction in sporulation efficiency. pXO1 also carries a gene encoding an Hfq-like protein, pXO1-137. Loss of pXO1-137 results in significant growth defects and reductions in toxin gene expression only when grown under toxin inducing conditions. Similarly, loss of a small RNA on pXO1, sRNA-1, results in similar growth defects and reductions in toxin gene production. Both increased and decreased expression of pXO1-137 and sRNA-1 result in growth defects suggesting narrow functional set points for Hfq and sRNA levels.

Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Tiruvoor G. Nagaraja / Cattle are a reservoir for major Shiga toxin-producing Escherichia coli (STEC), which includes STEC O157 and the top six non-O157 serogroups (STEC-6; O26, O45, O103, O111, O121, O145). Collectively known as the STEC-7, these organisms are harbored in the hindgut and shed in the feces of cattle, which can contaminate hides. The de-hiding step during beef cattle processing can introduce fecal contaminants from the hide onto the carcass surface, creating the potential for contaminated beef products. The STEC-7 have been declared by the USDA-Food Safety and Inspection Service as adulterants in ground beef and non-intact beef products, and are monitored during beef cattle processing. However, many of the culture- and PCR-based tests for detection and/or quantification of the STEC, particularly of the STEC-6, are not established or require improvement and also virulence characteristics of STEC strains from cattle have not been fully analyzed. Therefore, the following studies were conducted: 1. Immunomagnetic separation (IMS)-based culture-method for detection of STEC-6 in cattle feces was developed and compared to a PCR-based method; 2. Detection sensitivity of pooled vs. individual IMS beads for isolation STEC-6 from cattle feces was evaluated; 3. Real-time PCR assay, based on the clustered regularly interspaced short palindromic repeat sequence polymorphisms (CRISPR), was developed and validated for serotype-specific detection and quantification of STEC O157:H7 in cattle feces; 4. Virulence gene profiles of bovine enterohemorrhagic (EHEC), enteropathogenic (EPEC) and putative non-pathotype E. coli O103 strains were examined with whole genome sequence (WGS)-based comparative analysis; 5. Prevalence and concentration of STEC-7 of fed-beef, cull beef and cull dairy cattle were determined. The culture and PCR methods detected all six serogroups in samples negative by the other method. Based on noninferiority tests, detection with pooled IMS beads was not inferior to detection with individual beads. Detection limits of the CRISPR-based qPCR assay for cattle feces spiked with pure cultures were 2.1 x 10³ and 2.3 x 10⁰ colony-forming units/g before and after enrichment, respectively. WGS-based analysis of E. coli O103 strains revealed key differences in the virulomes and mobilomes of EHEC, EPEC, and putative non-pathotype strains. The prevalence study revealed that a significantly higher (P < 0.01) proportion of hide samples from fed beef cattle (4.8%) were positive for STEC O157:H7, compared to samples from cull beef (1.6%) or cull dairy (0.2%); the majority of quantifiable STEC O157:H7 from each cattle type was at concentrations between 3 to 4 log CFU/100 cm². These data contribute to a knowledge gap on prevalence and concentration of STEC-7 and surrogate bacteria on cattle hides and carcasses, respectively. Furthermore, the development and refinement of culture- and PCR-based screening assays may lead to increased surveillance of major STEC serogroups, especially if the potential of WGS-based comparative genomics in identifying novel gene targets can be harnessed.

This study has brought an overview of botulinum toxin and its influence on the human muscles, especially on spastic muscles. In the practical part is resumed experience with botulinum toxin A therapy in children with cerebral palsy. There were observed two groups. Group A (n=9) was measured before and after therapy. Patients in group B (n=24) filled out a table of the global spasticity scale, which they returned by mail. All the patients improved after the treatment. Powered by TCPDF (www.tcpdf.org)

Master of Science in Biomedical Sciences / Department of Diagnostic Medicine/Pathobiology / Weiping Zhang / Heat-labile enterotoxin (LT) is one of the most important toxins produced by enterotoxigenic Escherichia coli (ETEC). It consists of one A subunit (LTA) for intracellular enzymatic activity and five B subunits (LTB) forming a pentamer for binding to host cell receptors. In the last few decades, LT has been extensively studied as a strong immune stimulator, as well as an effective adjuvant with multiple immunomodulatory properties. To understand better the features of LT, we mapped B-cell linear epitopes of the enzymatic A subunit and explored the relationship between these epitopes and the toxicity of LT. Eleven B-cell linear (continuous) epitopes were in silico identified based on online software. In part one of the study, all 11 epitopes were fused into a modified ovalbumin carrier protein respectively. Each recombinant fusion protein was expressed and purified, and was characterized in ELISA and Western Blot using the anti-LT serum. Moreover, each fusion protein was used to immunize mice to determine immune response specific to LT in vivo. A total of eleven epitopes were identified from the LTA subunit. Results showed that anti-LT serum recognized all 11 epitopes, while the mouse immunization study indicated that antibodies derived from epitope 7 (₁₀₅SPHPYEQEVSA₁₁₅) had significantly greater anti-LT antibody titers and neutralized LT enterotoxicity more efficiently than the other epitopes. In part two of the study, to test whether individual epitope plays a role in LT toxicity, 10 epitopes in the A1 domain of LTA subunit were replaced by a foreign peptide respectively and the mutant LTs were examined for enterotoxicity. Data indicated that all these LT mutants showed enterotoxicity abolished. However, these LT mutants formed holotoxin structure and bound to GM1 in vitro. Results from this study indicated that replacement of these LT epitopes did not affect the forming of LT holotoxin structure and the binding to host receptors, indicating LT can serve as a safe vaccine platform to carry foreign antigens. With the immunodominant epitope 7 being kept while other LTA epitopes replaced by epitopes from other ETEC virulence factors, this platform can be used to construct broadly protective multivalent mucosal vaccines against ETEC, and perhaps as a universal platform for vaccines against other enteric diseases.

No description available.

Streptococcus pneumoniae (pneumococcus) is a major human pathogen and commensal organism of the nasopharynx. A major virulence factor of the pneumococcus is the cholesterol dependent, pore forming cytolysin pneumolysin. This toxin acts extracellularly, but the mechanism of release has not been well elucidated. Despite being a catalase negative organism, the pneumococcus produces up to millimolar concentrations of hydrogen peroxide through the activity of pyruvate oxidase. In all strains analyzed, deletion of the pyruvate oxidase gene yielded a significant reduction in the amount of PLY observed in the supernatant via western blot. A single strain, WU2 was also observed to have a significant (p<.05) reduction in the amount of PLY observed in the supernatant when treated with extracellular catalase. Furthermore, a significant correlation between hydrogen peroxide production and PLY release was observed in a panel of 15 clinical isolates.

No description available.

Indiana University-Purdue University Indianapolis (IUPUI) / Clostridium perfringens is an important human pathogen known to cause a range of diseases including diarrhea, necrotizing bowel disease and gas gangrene. Though potentially pathogenic, this microorganism is commonly identified in the fecal microbiota of healthy individuals. The major clinical findings associated with C. perfringens diseases are linked to production of potent toxins. In 1997, Gibert et al. identified a new toxin, the beta2 toxin, from a C. perfringens strain from a piglet with necrotic enteritis. Subsequently, this new beta2 toxin gene (cpb2) has been identified in C. perfringens from dogs, horses, and other animals. The principal objective of this investigation was to study cpb2 and the beta2 toxin in C. perfringens isolates from human sources. The C. perfringens isolates were grouped into three different populations: 1) fecal samples from patients suspected of having C. perfringens gastrointestinal illnesses (e.g. antibiotic-associated diarrhea or colitis), 2) extraintestinal specimen sources (e.g. wounds, abscesses, blood cultures), 3) a control group of isolates from healthy volunteers. Results of studies using different PCR methods and nucleotide sequencing revealed that cpb2 was present in the genome of isolates from all populations, and that the genetic variation between cpb2 from the different C. perfringens isolates was greater then expected. Using western blotting techniques, it was found that the beta2 protein was not expressed by all cpb2 positive C. perfringens isolates. Finally, different variants of cpb2 were cloned into E. coli, and the recombinant beta2 protein used in cell cytotoxicity assays. Results from these assays demonstrated that recombinant beta2 proteins caused a range of cellular damage at different levels of protein concentration and different lengths of time. Our results from these experiments provided new information regarding cpb2 in C. perfringens isolates from human sources; as well as on the range of variation of cpb2 genes, differences in beta2 toxin expression, and differences in the effects of recombinant beta2 toxin on enterocytes. This information could help to explain differences in virulence between C. perfringens isolates, differences in diseases and disease severity.

Yes / The Ad Hoc Group (AHG) of the States Parties to the Biological and Toxin Weapons Convention (BTWC) have touched from time to time on the question of the organisation needed to implement the legally binding instrument being negotiated to strengthen the BTWC. Now that the work of the AHG has intensified with the fleshing out of a rolling text for the legally binding instrument, the nature of the organisation is receiving more and more attention as its size and cost are likely to influence the nature and effectiveness of the regime developed by the AHG. This Briefing Paper considers what can be learned from existing relevant organisations, notably the World Health Organisation (WHO) and its counterparts for animal and plant diseases (OIE and FAO), the United Nations Special Commission (UNSCOM) on Iraq and the Organization for the Prohibition of Chemical Weapons (OPCW). The developments thus far in the AHG deliberations are then addressed and some estimates are made for the optimum size and cost of a BTWC rganisation. It is emphasised that these estimates are necessarily broad as the actual size of the BTWC Organization will depend on the precise functions and responsibilities that it is given.

Yes / The Ad Hoc Group (AHG) of the States Parties to the Biological and Toxin Weapons Convention (BTWC) has the consideration of measures to implement Article X of the Convention as an element of its mandate agreed by the Special Conference in September 1994. The AHG has considered how to address this at each of its substantive meetings with a Friend of the Chair, initially Ambassador Jorge Berguno of Chile and subsequently, Carlos Duarte of Brazil carrying out this responsibility. As progress is being made on the development of the rolling text for the Protocol to strengthen the Convention, it is timely to consider how the implementation of Article X might contribute to the strengthening of the effectiveness of the Convention. Briefing Paper No 6 considered some of the developments that have occurred nationally, regionally and internationally in respect of the use of bacteriological (biological) agents and toxins for peaceful purposes. It noted that there is increasing awareness world-wide because of public health and environmental concerns of the need to control the handling, use, storage and transfer of such biological agents. That paper examined some of the current controls and regulations for biosafety and the international initiatives that are ongoing to strengthen biosafety around the world. These were seen as building blocks which might be considered from a point of view of strengthening the BTWC as well as contributing to the implementation of Article X although care will need to be taken in the Protocol for the AHG to avoid unnecessary duplication with other international activities. This Briefing Paper is complementary to Briefing Paper No 6 as it considers the national regulations in the UK, the EEC and in the United States as well as some other countries in respect of micro-organisms with the aim of providing some further building blocks to be considered in the strengthening of the BTWC and the implementation of Article X of the Convention. The challenging goal continues to be to identify how these other national, regional and international activities can be utilised to contribute to the strengthening of the BTWC.