Publications

4498

AbstractBackground:  “Candidatus Helicobacter heilmannii” induce chronic gastritis, which eventually leads to gastric B‐cell type mucosa‐associated lymphoid tissue (MALT) lymphoma. This study was performed using an animal model of infection with “Candidatus Helicobacter heilmannii” to elucidate how this chronic inflammation is induced or maintained.Materials and Methods:  BALB/c mice were infected with the “Candidatus Helicobacter heilmannii” isolate SH4. The animals were examined at 8, 26, 54, and 83 weeks after the infection. The stomach of the animals was resected and immunostained for peripheral lymph node addressin (PNAd) and mucosal addressin cell adhesion molecule 1 (MAdCAM‐1), “Candidatus Helicobacter heilmannii,” and CD45R/B220. An in vitro binding assay with L‐ and E‐selectin·IgM chimeric proteins was performed. Real‐time polymerase chain reaction was used to evaluate transcripts of N‐acetylglucosamine‐6‐O‐sulfotransferases (GlcNAc6STs), which direct the expression of the PNAd and MAdCAM‐1.Results:  Chronic gastritis developed in the infected animals, and its severity increased with the duration of the infection. B‐cell type MALT lymphoma developed in some animals at 54 and 83 weeks after infection. PNAd‐ and MAdCAM‐1‐expressing high endothelial venule (HEV)‐like vessels were induced in infected animals which developed chronic gastritis and MALT lymphoma. The number of HEV‐like vessels increased as chronic inflammation progressed. The induced HEV‐like vessels were bound by L‐ and E‐selectin·IgM chimeric protein. mRNA expressions of GlcNAc6ST‐1 and MAdCAM‐1 increased in the infected animals.Conclusions:  HEV‐like vessels expressing GlcNAc6ST‐1‐mediated L‐selectin ligand carbohydrate and MAdCAM‐1 may play a crucial role in the pathogenesis of “Candidatus Helicobacter heilmannii”‐induced chronic gastritis and MALT lymphoma.

Five Gram-negative, facultatively anaerobic, non-spore-forming, coccoid rod-shaped bacterial isolates were obtained from infant formula and an infant formula production environment and were investigated by use of a polyphasic taxonomic study. Biochemical tests and partialrpoBgene sequence analysis of the five isolates revealed that they formed two distinct groups in the familyEnterobacteriaceae, closely related to several species of the generaPantoeaandErwinia, which indicated a phylogenetic position within the genusPantoeaor the genusErwinia. Multilocus sequence analysis of concatenated partialatpD,gyrB,infBandrpoBgene sequences of two of the isolates suggested that they represented two novel species of the genusPantoea, phylogenetically related most closely toPantoea septica. The five isolates had general characteristics consistent with those of the genusPantoea, and DNA–DNA hybridizations between two representatives and the type strains of their phylogenetically closest relatives based on comparative 16S rRNA gene sequence analysis showed that the isolates represented two novel genospecies. These two genospecies could be differentiated from each other based on fermentation of galacturonate, sorbitol and potassium 5-ketogluconate. They could be differentiated from phylogenetically relatedPantoeaspecies based on their ability to ferment lactose and to utilizeβ-gentiobiose and raffinose, their inability to ferment or utilized-arabitol, and their inability to produce indole. On the basis of the results obtained, the five isolates are considered to represent two novel species of the genusPantoea, for which the namesPantoea gaviniaesp. nov. (type strain A18/07T=LMG 25382T=DSM 22758T) andPantoea calidasp. nov. (type strain 1400/07T=LMG 25383T=DSM 22759T) are proposed.

Citrus huanglongbing, putatively caused by the associated bacterium ‘Candidatus Liberibacter asiaticus’, is the greatest threat to the world citrus industry today. The bacterium is spread locally and regionally by the citrus psyllid Diaphorina citri, and also can be disseminated by propagation of contaminated scion budwood that is grafted to the appropriate rootstock. The planting of ‘Ca. Liberibacter asiaticus’-free trees is a component of a comprehensive strategy to manage huanglongbing. In contrast to the scion budwood, the rootstocks used to produce these trees are grown from seed. This research was undertaken to provide evidence as to whether or not ‘Ca. L. asiaticus’ can be transmitted through seed. Two groups of 360 or more seedlings each of various citrus species were grown from seed removed from fruit on trees that were symptomatic for huanglongbing and confirmed to be infected with ‘Ca. L. asiaticus’ by polymerase chain reaction (PCR) tests. These seedlings were tested multiple times over periods of up to 3 years. No symptoms typical of huanglongbing, such as blotchy leaf mottle, chlorotic shoots, or dieback of branches, were observed in these seedlings, and none of these 723 seedlings tested positive for the presence of ‘Ca. L. asiaticus’ even after repeated testing by sensitive quantitative PCR assays. Some sour orange seedlings did have quite pronounced and atypical growth, including stunting and mild to severe leaf malformation. These atypical growth habits were limited to seedlings that arose from zygotic embryos as determined by expressed-sequence tag simple-sequence repeat analyses. Thus, no evidence of transmission of ‘Ca. L. asiaticus’ via seed was obtained, and an earlier report of transmission of the pathogen through seed was not confirmed.

Crotalaria spectabilis Roth. (Fabaceae), commonly known as showy rattlebox, is an herbaceous legume mainly used as a green manure crop to improve soil properties and as a source of durable fiber. However, the plant is toxic to mammals and birds because of the presence of pyrrolidizine alkaloids. A native of India and the Malay Peninsula, the species has been introduced into other areas such as the United States and Pacific Islands where the plant is an invader of cultivated lands. Fasciated rattlebox plants were sighted in fields in New Delhi in February 2010, with approximately 99% of the examined plants symptomatic. Symptoms included flattening of stems in a descending gradient of severity from the apex to the base of each affected branch. Shoots showed longitudinal undulations bearing highly reduced leaves and uneven distribution of flowers and fruits. To identify the causal agent, 10 symptomatic plants and 8 asymptomatic plants (latter sampled from a field approximately 1.5 km away) were collected for nucleic acid analysis. Total genomic DNA was extracted from flattened stems as well as the roots of symptomatic plants, and from the same tissues of asymptomatic plants, and subjected to nested-PCR using phytoplasma 16S ribosomal DNA universal primer pair P1/P7, followed by R16F2/R2 (4). A known aster yellows-infected Catharanthus roseus plant was used as a control sample. Results depicted a characteristic phytoplasma amplicon of 1.25 kb in all samples from symptomatic plants and the control plant. No amplification was observed from asymptomatic plants. To obtain a full-length sequence, a representative amplicon was purified with the QIAquick gel extraction kit (QIAGEN, Valencia, CA), cloned into pGEM-T Easy vector (Promega, Madison, WI), and sequenced. Comparison of the 1,243-bp sequence (Genbank Accession No. HM137557) using BLAST analysis of the NCBI database showed 99% homology with sequences of members of 16SrI group phytoplasmas, i.e., ‘Candidatus Phytoplasma asteris’ such as Japanese spurge yellows (AB551736.1), Mulberry yellow dwarf (GQ249410.1), and Bamboo witches'-broom (FJ853161.1) phytoplasmas. The profiles of in vitro restriction fragment length polymorphism (RFLP) analysis obtained by digestion of the nested-PCR products with HhaI, KpnI, and AluI were similar to those of in silico RFLP analyses and coincided with the pattern of the 16SrI-B subgroup. Phylogenetic analysis of phytoplasma 16S rDNA sequences based on the maximum likelihood method using MEGA Version 4.1 also placed the Crotalaria fasciation (CF) phytoplasma within the 16SrI-B cluster. In India, C. tetragona plants infected with 16SrI phytoplasma (FJ185141) causing witches'-broom symptoms (1) showed 98% similarity with the CF phytoplasma. However, the results support previous molecular investigations associating 16SrI phytoplasma with fasciation of herbaceous plants, including Lilium spp. (2) and Asparagus officinalis (3). To our knowledge, C. spectabilis represents a new host that can be fasciated as a result of phytoplasma infection. Because of the weedy nature of C. spectabilis, this host could facilitate spread of the phytoplasma. References: (1) P. Baiswar et al. Plant Pathol. 19:17, 2009. (2) A. Bertaccini et al. FEMS Microbiol. Lett. 249:79, 2005. (3) J. Franova and K. Petrzik. J. Phytopathol. 158:317, 2010.K. (4) D. E. Gundersen and I.-M. Lee. Phytopathol. Mediterr. 35:144, 1996.