Inactivation of SAM-methyltransferase is the mechanism of attenuation of a historic louse borne typhus vaccine strain

Louse borne typhus (also called epidemic typhus) was one of man's major scourges, and epidemics of the disease can be reignited when social, economic, or political systems are disrupted. The fear of a bioterrorist attack using the etiologic agent of typhus, Rickettsia prowazekii, was a reality. An attenuated typhus vaccine, R. prowazekii Madrid E strain, was observed to revert to virulence as demonstrated by isolation of the virulent revertant Evir strain from animals which were inoculated with Madrid E strain. The mechanism of the mutation in R. prowazekii that affects the virulence of the vaccine was not known. We sequenced the genome of the virulent revertant Evir strain and compared its genome sequence with the genome sequences of its parental strain, Madrid E. We found that only a single nucleotide in the entire genome was different between the vaccine strain Madrid E and its virulent revertant strain Evir. The mutation is a single nucleotide insertion in the methyltransferase gene (also known as PR028) in the vaccine strain that inactivated the gene. We also confirmed that the vaccine strain E did not cause fever in guinea pigs and the virulent revertant strain Evir caused fever in guinea pigs. We concluded that a single nucleotide insertion in the methyltransferase gene of R. prowazekii attenuated the R. prowazekii vaccine strain E. This suggested that an irreversible insertion or deletion mutation in the methyl transferase gene of R. prowazekii is required for Madrid E to be considered a safe vaccine

Genotyping Rickettsia prowazekii Isolates

We developed a typing method that can differentiate 8 strains of Rickettsia prowazekii into 7 genotypes. This method can be used to type and trace the origin of R. prowazekii isolated from samples collected during epidemics after a bioterrorism attack

Wolbachia Prophage DNA Adenine Methyltransferase Genes in Different Drosophila-Wolbachia Associations

Wolbachia is an obligatory intracellular bacterium which often manipulates the reproduction of its insect and isopod hosts. In contrast, Wolbachia is an essential symbiont in filarial nematodes. Lately, Wolbachia has been implicated in genomic imprinting of host DNA through cytosine methylation. The importance of DNA methylation in cell fate and biology calls for in depth studing of putative methylation-related genes. We present a molecular and phylogenetic analysis of a putative DNA adenine methyltransferase encoded by a prophage in the Wolbachia genome. Two slightly different copies of the gene, met1 and met2, exhibit a different distribution over various Wolbachia strains. The met2 gene is present in the majority of strains, in wAu, however, it contains a frameshift caused by a 2 bp deletion. Phylogenetic analysis of the met2 DNA sequences suggests a long association of the gene with the Wolbachia host strains. In addition, our analysis provides evidence for previously unnoticed multiple infections, the detection of which is critical for the molecular elucidation of modification and/or rescue mechanism of cytoplasmic incompatibility

Evidence of a Louse-Borne Outbreak Involving Typhus in Douai, 1710-1712 during the War of Spanish Succession

Background: The new field of paleomicrobiology allows past outbreaks to be identified by testing dental pulp of human remains with PCR. Methods: We identified a mass grave in Douai, France dating from the early XVIII th century. This city was besieged during the European war of Spanish succession. We tested dental pulp from 1192 teeth (including 40 from Douai) by quantitative PCR (qPCR) for R. prowazekii and B. quintana. We also used ultra-sensitive suicide PCR to detect R. prowazekii and genotyped positive samples. Results and Discussion: In the Douai remains, we identified one case of B. quintana infection (by qPCR) and R. prowazekii (by suicide PCR) in 6/21 individuals (29%). The R. prowazekii was genotype B, a genotype previously found in a Spanish isolate obtained in the first part of the XX th century. Conclusion: Louse-borne outbreaks were raging during the XVIII th century; our results support the hypothesis that typhus was imported into Europe by Spanish soldiers from America

Bioterrorism

This book consists of nine chapters, written by international authorities, discussing various aspects of bioterrorism preparedness and response. Five of the chapters are agent-specific and highlight the pathogenesis, prevention and treatment, and the potential of specific organisms (Rickettsia and Yersinia pestis) or toxins (ricin, botulinum neurotoxins, and staphylococcal enterotoxins) to be used for nefarious purposes. Four chapters discuss different aspects of detecting and responding to a bioterrorism attack. These include methods for spatio-temporal disease surveillance, international laboratory response strategies, detection of botulinum neurotoxins in food and other matrices, and the use of physical methods (ie Raman spectroscopy) to detect spores

Molecular characterization of cytoplasmicnuclear Male sterillty (cms) sources and Tallidwarf near-isocenic lines in pearl millet

The conventional method of classifying CMS lines on fertility restoration atterns is cumbersome and time consuming. Restriction fragment length polymorphism (RFL P ) of mitochondria1 (mt) DNA provides a rapid and effective method to assess heterogeneity among male-sterile cytoplasms. With this objective, six isonuclear A-lines (81A, with Tift 23A, cytoplasm, ICMA 88001 (= 81 A,) with violaceum cytoplasm, 81 A (= 81 A ) with monodii = violaceum cytoplasm, Pb 310A, and Pb 31 1A, with A, cytoplasm, a n $ ~ b4 06a with A cytoplasm), nine male-sterile lines from Large-seeded Genepool ( LSGP 6, LSGP 14, ~ G17P, LS GP 22, LSGP 28, LSGP 36, LSGP 43, LSGP 55 and LSGP 66) and two CMS lines each from Early Genepool (EGP 1 and EGP 2) and Population Varieties (PV 1 and PV 2) were characterized for variation in their mitochondrial genomes following Southern blot hybridizations using homologous (pearl millet 13.6 kb, 10.9 kb, 9.7 kb and 4.7 kb clones) and heterologous (maize atp6 and cox1 clones) mtDNA probes. Based on RFLP banding pattern we identified seven cytoplasmic groups from the LSGP. Of the LSGP cytoplasms, LSGP 43, LSGP 55 and LSGP 66 were most diverse.....