Publications

4467

Abstract: Phylogenetic analysis of five rickettsial genes (17‐kDa gene, gltA, ompB, ompA, and sca4) from two molecular isolates of Candidatus Rickettsia andeanae from two ticks (Amblyomma maculatum and Ixodes boliviensis) collected from two domestic horses living in two separate locations in northern Peru (Coletas and Naranjo) was conducted to more clearly characterize this recently reported novel spotted fever group (SFG) rickettsia. Following nested polymerase chain reaction (PCR) amplification of the17‐kDa gene, gltA, ompB, ompA, and sca4, amplicons were purified, sequenced, and compared to those downloaded from GenBank. Phylogenetic analyses of the Candidatus Rickettsia andeanae sequences generated from17‐kDa gene (483 bp), gltA (1185 bp), ompA (1598 bp), ompB (4839 bp), and sca4 (2634 bp) demonstrated that they aligned strongly with those of SFG rickettsiae. Moreover, the sequences of these five genes most closely aligned with the following rickettsiae: ompA: Rickettsia sp RpA4 (98.03%), R. sp DnS28 (97.90%), and R. rhipicephali and R. massiliae (97.11%); ompB: R. aeschlimannii (97.22%), R. rhipicephali (97.20%), and R. sp Bar 29 (97.10%); and sca4: R. massiliae (97.8%), R. rhipicephali, and R. slovaca (97.7%). These results from the additional phylogenetic analyses of Candidatus Rickettsia andeanae confirm its inclusion within, and distance and uniqueness from, other known SFG rickettsiae.

ABSTRACT Huanglongbing (yellow dragon disease) is a destructive disease of citrus. The etiological agent is a noncultured, phloem-restricted alpha-proteobacterium, “ Candidatus Liberibacter africanus” in Africa and “ Candidatus Liberibacter asiaticus” in Asia. In this study, we used an omp -based PCR-restriction fragment length polymorphism (RFLP) approach to analyze the genetic variability of “ Ca . Liberibacter asiaticus” isolates. By using five different enzymes, each the 10 isolates tested could be associated with a specific combination of restriction profiles. The results indicate that the species “ Ca . Liberibacter asiaticus,” even within a given region, may comprise several different variants. Thus, omp -based PCR-RFLP analysis is a simple method for detecting and differentiating “ Ca . Liberibacter asiaticus” isolates.

ABSTRACT “ Helicobacter heilmannii ” (proposed name) type 1 colonizes the human stomach. It has been shown to be identical to“ Candidatus Helicobacter suis,” a Helicobacter species colonizing the stomachs of >60% of slaughter pigs. This bacterium has not been isolated in vitro until now. Antibiotic susceptibility testing of “ Candidatus Helicobacter suis” has not been carried out so far. For the present study, a mouse model was adopted to evaluate the antibiotic susceptibility of this organism. Mice infected with“ Candidatus Helicobacter suis” were treated with amoxicillin and omeprazole, a therapy which is used to treat H. heilmannii infections in humans. Two different isolates of“ Candidatus Helicobacter suis” were tested. The excretion of bacterial DNA was assessed during treatment, using PCR on fecal samples. At the end of the experiment, 8 days after the cessation of treatment, the presence of infection was evaluated using a urease test and a PCR test on stomach samples. A marked decrease in the excretion of bacterial DNA was observed a few days after the onset of treatment, and the level remained low until the end of the experiment. A difference in susceptibility between the two“ Candidatus Helicobacter suis” isolates was pointed out. The in vivo mouse model infected with“ Candidatus Helicobacter suis” will be useful for further screening of potential therapeutic regimens.

During 2003, surveys of sugarcane yellow leaf disease and papaya bunchy top-like disease were carried out on plantations in Havana province, Cuba, to determine the roles of weeds and Auchenorrhyncha insects in the epidemiology of these diseases. More than 250 plant and insect samples were collected and indexed by using a nested PCR for phytoplasma 16S rDNA with the generic primer pairs P1/P7 and R16F2n/R16R2. The PCR products were further characterized by restriction fragment length polymorphism using HaeIII, AluI, Sau3AI, Tru9I, HhaI, HpaII and TaqI endonucleases, giving patterns that distinguished them from those of the other reference phytoplasmas analysed. Phylogenetic analysis of 16S rRNA gene sequences identified the phytoplasmas present in sugarcane (Saccharum officinarum L.), Cynodon dactylon L., Conyza canadensis L. Cronq., Sorghum halepense L. Pers., Macroptilium lathyroides L. Urb., Saccharosydne saccharivora (Westwood) and Cedusa spp., and those in papaya (Carica papaya L.) and Empoasca papayae, as two novel provisional phytoplasma species. We propose that these phytoplasmas should be given Candidatus status, as ‘Candidatus Phytoplasma graminis' and ‘Candidatus Phytoplasma caricae’, respectively.

A novel anaerobic, thermophilic, alkalitolerant bacterium, strain 2204T, was isolated from a hot spring of the Baikal Lake region. The cells of strain 2204Twere straight rods of variable length, Gram-positive with an S-layer, motile with one to two lateral flagella, and often formed aggregates of 3–15 cells. The isolate was shown to be an obligate anaerobe oxidizing CO and producing equimolar quantities of H2and CO2according to the equation CO+H2O→CO2+H2. No organic substrates were used as energy sources. For lithotrophic growth on CO, 0·2 g acetate or yeast extract l−1was required but did not support growth in the absence of CO. Growth was observed in the temperature range 37–68 °C, the optimum being 55 °C. The pH range for growth was 6·7–9·5, the optimum pH being 8·0. The generation time under optimal conditions was 1·3 h. The DNA G+C content was 45 mol%. Penicillin, erythromycin, streptomycin, rifampicin, vancomycin and tetracycline completely inhibited both growth and CO utilization by strain 2204T. Thus, isolate 2204Twas found to be the first known moderately thermophilic and alkalitolerant H2-producing anaerobic carboxydotroph. The novel bacterium fell within the cluster of the familyPeptococcaceaewithin the low-G+C-content Gram-positive bacteria, where it formed a separate branch. On the basis of morphological, physiological and phylogenetic features, strain 2204Tshould be assigned to a novel genus and species, for which the nameThermincola carboxydiphilagen. nov., sp. nov. is proposed. The type strain is strain 2204T(=DSM 17129T=VKM B-2283T=JCM 13258T).

The obligately intracellular coccoid bacterium UWE25, a symbiont of Acanthamoeba spp., was previously identified as being related to chlamydiae based upon the presence of a chlamydia-like developmental cycle and its 16S rRNA gene sequence. Analysis of its complete genome sequence demonstrated that UWE25 shows many characteristic features of chlamydiae, including dependency on host-derived metabolites, composition of the cell envelope and the ability to thrive as an energy parasite within the cells of its eukaryotic host. Phylogenetic analysis of 44 ribosomal proteins further confirmed the affiliation of UWE25 to the ‘Chlamydiae’. Within this phylum, UWE25 could be assigned to the family Parachlamydiaceae based on comparative analyses of the 16S rRNA, 23S rRNA and endoribonuclease P RNA genes. The distinct dissimilarities from its closest relative, Parachlamydia acanthamoebae Bn9 T (7·1, 9·7 and 28·8 %, respectively), observed in this analysis justify its classification in a new genus. Therefore, the name ‘Candidatus Protochlamydia amoebophila’ is proposed for the designation of the Acanthamoeba sp. symbiont UWE25 (=ATCC PRA-7).