16S-23S Spacer Research Articles

‘Candidatus Phytoplasma balanitae’ associated with witches’ broom disease of Balanites triflora

A phytoplasma was identified in naturally infected wild Balanites triflora plants exhibiting typical witches' broom symptoms (Balanites witches' broom: BltWB) in Myanmar. The 16S rRNA gene sequence revealed that BltWB phytoplasma had the highest similarity to that of 'Candidatus Phytoplasma ziziphi' and it was also closely related to that of 'Candidatus Phytoplasma ulmi'. Phylogenetic analysis of the 16S rRNA gene sequences indicated that the BltWB phytoplasma clustered as a discrete subclade with Elm yellows phytoplasmas. RFLP analysis of the 16S rRNA gene including the 16S-23S spacer region differentiated the BltWB phytoplasma from 'Ca. P. ziziphi', 'Ca. P. ulmi' and 'Candidatus Phytoplasma trifolii'. Analysis of additional ribosomal protein (rp) and translocase protein (secY) gene sequences and phylogenetic analysis of BltWB showed that this phytoplasma was clearly distinguished from those of other 'Candidatus Phytoplasma' taxa. Taking into consideration the unique plant host and the restricted geographical occurrence in addition to the 16S rRNA gene sequence similarity, the BltWB phytoplasma is proposed to represent a novel taxon, 'Candidatus Phytoplasma balanitae'.

'Candidatus Phytoplasma cynodontis' associated with white leaf disease of golden beard grass (Chrysopogon acicalatus)

A phytoplasma was detected in golden beard grass (Chrysopogon acicalatus) displaying white leaf symptoms near vegetative fields at the Tatkone region in Myanmar, 2011. Based on restriction fragment length polymorphism analysis of PCR-amplified 16S rRNA genes, including the 16S-23S spacer region and part of the 23S rRNA gene, the phytoplasma was identified as a member of the Bermuda grass white leaf phytoplasma (BGWL) group. The golden beard grass white leaf phytoplasma (GBGWL) 16S rRNA gene sequence exhibited over 98.7% similarity with all members of BGWL group phytoplasmas and 99.5% similarity with Thailand Bermuda grass white leaf phytoplasma (AF248961). In addition, the GBGWL phytoplasma was confirmed to be a member of 'Candidatus Phytoplasma cynodontis' by phylogenetic analyses.

Molecular Identification of Rare Clinical Mycobacteria by Application of 16S-23S Spacer Region Sequencing.

In addition to several molecular methods and in particular 16S rDNA analysis, the application of a more discriminatory genetic marker, i.e., 16S-23S internal transcribed spacer gene sequence has had a great impact on identification and classification of mycobacteria. In the current study we aimed to apply this sequencing power to conclusive identification of some Iranian clinical strains of mycobacteria. The test strains consisted of nineteen mycobacterial isolates which were initially identified by the use of conventional phenotypic techniques and molecular methods and subjected to further definitive identification using the 16S-23S internal transcribed spacer gene sequencing. Out of 19 studied strains, 7 isolates were found to be rapidly growing and 12 isolates as slowly growing mycobacteria. With the exception of one isolate, i.e., the isolate HNTM87, which yielded a distinct ITS sequence incomparable with all previously identified mycobacteria, the remaining isolates produced the sequences similar to the established mycobacteria and were clearly identified and differentiated from closely related taxa. A phylogenetic tree based on maximum parsimony analysis of 16S-23S internal transcribed spacer gene sequences constructed showing the relatedness of Iranian clinical isolates with the closely related type species of mycobacteria. This study showed that the 16S-23S internal transcribed spacer gene of the genus Mycobacterium exhibits a high variation which is of value for discriminating closely related taxa and could be used independently or in combination with 16S rDNA sequencing to delineate the true identity of rare mycobacterial species.

Final Results of a Multicenter Phase II Trial with Translational Elements to Investigate the Possible Infective Causes of Ocular Adnexal Marginal Zone B-Cell Lymphoma (OAMZL) with Particular Reference to Chlamydia Species and the Efficacy of Doxycycline As First-Line Lymphoma Treatment (the IELSG#27 TRIAL)

Abstract▪267▪This icon denotes a clinically relevant abstract BACKGROUND:Chlamydophila psittaci (Cp) infection carries a potential pathogenic role in OAMZL and has been detected in tumor tissue of a high proportion of patients (pts), with geographic differences in its prevalence rates. The use of immunohistochemistry, immunofluorescence and electronic microscopy techniques has identified monocyte/macrophage as carriers of Cp in OAMZL, and bacteria eradication with doxycycline (DOXY) has been associated with lymphoma regression in half of pts. Notwithstanding these achievements, clinical and therapeutic knowledge on this association mainly result from retrospective studies or trials including also patients with relapsed disease after wide-ranging follow-up. Prospective studies focusing exclusively on pts with newly diagnosed OAMZL do not exist. Herein, we report the final results of the first international prospective phase II trial aimed to investigate Cp prevalence and DOXY efficacy as first-line therapy in pts with newly diagnosed OAMZL (ClinicalTrial.gov NCT01010295). AIMS:To elucidate prevalence of Chlamydiae infections, and to evaluate the efficacy of DOXY both in terms of bacterial eradication and as upfront anti-lymphoma therapy in pts with newly diagnosed OAMZL. METHODS:This trial included two different parts, named A and B. The part A included pts with newly diagnosed stage-IEA OAMZL and measurable/parametrable disease; these pts were assessed for chlamydial infection and were treated with DOXY 100 mg orally twice daily for 21 days. The part B included pts with lymphoma categories other than MZL, non neoplastic lesions of the ocular adnexae or OAMZL not eligible for part A; these pts were treated following local guidelines.Chlamydial infections were assessed on diagnostic tumor samples, conjunctival swabs and peripheral blood mononuclear cells (PBMC) from A and B pts by three different PCR protocols targeting 16rRNS and intergenic spacer 16S–23S, ompA and hsp60, respectively. Bacterial eradication was tested on conjunctival swabs and PBMC collected at basal time, at 3 and 12 months after DOXY (only part A pts). Clinical and ophthalmologic examination and MRI imaging were performed at the same timepoints, and every six months during follow-up. RESULTS:From August 2006 to November 2010, 54 pts from 6 centers were enrolled; 34 pts with OAMZL entered the part A, while 13 pts with non-parametrable OAMZL and 7 pts with other lymphoproliferative disorders entered the part B. Diagnostic material was available for molecular analysis in 44 cases; Cp was detected in biopsies of 32/37 OAMZL (86%) and in 4/7 non-MZL. All cases were negative for C. pneumoniae and C. trachomatis. Among 36 Cp+ pts, infection was detected in 100% of swabs and in 75% of PBMC; swabs or PBMCs from pts with Cp-negative OAMZL were invariably negative.All pts completed the planned DOXY treatment; no relevant toxicity was reported. Among 34 pts entering the part A, 28 were evaluable for bacteria eradication (Cp detected in swabs in 8 pts, in PBMC in one, in both samples in 19). At 3 months from DOXY, Cp was not detected in swabs and PBMC of 14 evaluable pts, with an eradication rate of 50%; at one year of follow-up, Cp was still detected in three of these apparently eradicated pts.Lymphoma regression after DOXY treatment (part A) was complete in six pts (18%; 95%CI: 5–31%), and partial in 16 (ORR= 65%, 95%CI: 49–81%); 11 had SD and one PD. At a median follow-up of 27 months (range 3–51), 13 pts experienced relapse, with a 2-year PFS of 55±9%. A trend to a higher response rate (82% vs. 53%; p=0.12) and 3-yr PFS (72% vs. 52%; p=0.14) was observed in eradicated pts. No significant association between site of presentation (conjunctiva vs. orbit) and clinical outcome was detected. There was no association between animal exposure and bacteria eradication or lymphoma response. All patients but two (stroke and myocardial infarction) are alive, with a 3-yr OS of 92%. CONCLUSIONS:Cp infection is frequent in newly diagnosed OAMZL, and could be also present in other lymphoproliferative disorders of the ocular adnexae. First-line DOXY is an active, safe and rational strategy in pts with limited-stage OAMZL, with lymphoma regression in 65% of cases. However, DOXY failed to eradicate Cp infection in half of pts, with a negative impact on outcome. Further investigations aimed to identify additional potential infective agents associated with OAMZL and more active antibiotic regimens are warranted. Disclosures:Montalban:Red Temática de Investigación Cooperativa en Cancer (RETICC): Research Funding; Asociación Española contra el Cancer: Research Funding.

Genetic diversity in monophasic (1,4,[5],12:i:- and 1,4,[5],12:-:1,2) and in non-motile (1,4,[5],12:-:-) variants of Salmonella enterica S. Typhimurium

Monophasic variants of Salmonella enterica serotype Typhimurium, lacking either the second phase H-antigen with the antigenic formula 1,4,[5],12:i:- or lacking the first phase H-antigen with the antigenic formula 1,4,[5],12:-:1,2, and a non-motile variant lacking both flagellar phases were phenotypically and genotypically characterised. The genetic diversity of a collection of 58 isolates of Salmonella enterica 1,4,[5],12:i:- (n=23), 1,4,[5],12:-:1,2 (n=17), 1,4,[5],12:-:- (n=18) mainly from swine, bovine and poultry sources using a novel multiplex real-time PCR to simultaneously target different classes of genes (i.e. 10 loci) was investigated in regards to antimicrobial resistance and pulsed field gel electrophoresis (PFGE) typing. The majority of the S. 1,4,[5],12:i:- strains were genotypically confirmed to be variant of serotype Typhimurium, except two mdh negative isolates. Five fljB positive S. 1,4,[5],12:i:- variants were detected and could be considered as “inconsistent” variant. Moreover, presence of the fliC and fljB amplicons in the phenotypically monophasic S. 1,4,[5],12:-:1,2 and non-motile isolates suggest that their expressions should be blocked by mutation or deletion in the promoter regions linked to the flagellar phase expression. Based on the combination of detection of the following markers: Salmonella Pathogenic Islands (SPI-2 to SPI-5), virulence plasmid (spvC), antimicrobial resistance markers (blaTEM and sul1), Salmonella Genomic island (SGI-1 left junction), the integrase from class 1 integron (intI1) and DT104 16S-23S spacer region, thirteen different genotypes were identified among the 58 isolates. All investigated isolates showed virulence potential as determined by the presence of four SPI markers, which was exhibited in the genotype A9 mainly encountered for S. 1,4,[5],12:-:1,2 isolates. In contrast, high-frequency of markers were encountered for S. 1,4,[5],12:i:- assigned to genotypes B7 gathering almost all 10 markers except SGI-1. Frequencies of SGI-1 markers typically detected in serotype Typhimurium strains differed strongly among the three variants ranging from 0% for fljB negative S. 1,4,[5],12:i:- to 58.8% for S. 1,4,[5],12:-:-. The three variants were clearly phenotypically and genotypically distinct and varied in their resistance-types as well as in their PFGE types and genotypes newly described in this study. This first study on the French collection from non human sources could suggest presence of the Spanish clone in France as well as, to a lesser extent, some “inconsistent” variants exhibiting the resistance-type AM S SSS TE previously described in different European countries.

Mycobacterium heckeshornenseInfection in HIV-infected Patient

<i>Mycobacterium heckeshornense</i>Infection in HIV-infected Patient

Characterization of Strains of Xanthomonas axonopodis pv. dieffenbachiae from Bacterial Blight of Caladium and Identification of Sources of Resistance for Breeding Improved Cultivars

Bacterial blight, caused by Xanthomonas axonopodis pv. dieffenbachiae (Xad), is the most common foliar disease of caladium, an ornamental aroid grown for its colorful foliage. The disease can reduce the marketability of caladiums produced as potted plants and lower the yield of caladiums grown for tuber production. Three bacterial strains were isolated from symptomatic caladiums and identified as Xad using fatty acid analysis, carbon source use, and the sequence of the 16S-23S spacer, and tested for virulence against three susceptible cultivars. Two strains were virulent to all of the cultivars; however, one strain was differentially pathogenic, virulent against two cultivars, but not to the usually highly susceptible ‘Candidum’. In greenhouse inoculation tests of 17 cultivars and one breeding line, four cultivars were ranked as highly susceptible, nine as moderately susceptible, and five as resistant. Ten of these cultivars were also evaluated with natural infection in the field with good agreement between the results of the greenhouse and field evaluations. Cultivars White Queen, Florida Red Ruffles, Florida Sweetheart, Candidum Jr., and Mrs. Arno Nehrling have been identified as resistant to bacterial blight in greenhouse or field evaluations and can potentially be used in future breeding efforts to produce improved cultivars.

MOLECULAR CHARACTERIZATION AND TRANSMISSION OF BERMUDA GRASS WHITE LEAF PHYTOPLASMA IN IRAN

SUMMARY Symptoms of Bermuda grass white leaf (BGWL) were observed in several provinces of Iran. Among leafhoppers collected in BGWL-affected areas, Exitianus capicola tested positive for phytoplasma in PCR assays and transmitted the BGWL agent to healthy Bermuda grass plants. Using P1/P7 primer pair, target DNA fragments of approximately 1.8 kbp were amplified from diseased samples of Bermuda grass plants from 10 widely separated areas in six provinces. Restriction fragment length polymorphism (RFLP) analysis of P1/P7 PCR products identified two BGWL isolates from Firouzabad and Juyom (Fars province) denoted FBGWL and JBGWL as members of the Bermuda grass white leaf group, 16SrXIV. Percent homology and phylogenetic analysis of 16S rDNA and 16S-23S spacer region (SR) sequences showed also that FBGWL and JBGWL are related to the 16SrXIV group. Based on RFLP and phylogenetic analyses, FBGWL and JBGWL isolates were distinguishable from each other and were classified in subgroups 16SrXIV-A and 16SrXIV-B, respectively. This is the first report of BGWL disease and leafhopper transmission of the associated phytoplasma in Iran.

Detection of a New Bacterium Related to Xanthomonas fuscans subsp. aurantifolii Infecting Swingle Citrumelo in Brazil.

In March 2009, in a sweet orange orchard (Citrus sinensis) cv. Valencia grafted on Swingle citrumelo (C. paradisi Macf. × Poncirus trifoliata L. Raf.) rootstock in Severínia County, São Paulo State, Brazil, approximately 40 trees were detected with small, necrotic, dark brown leaf spots. These lesions occurred whether or not citrus leafminer (Phyllocnistis citrella) was present and they were only found on leaves from branches arising from the rootstock. Sweet orange foliage was not affected even when in contact with infected rootstock branches. Symptoms were unusual and distinct from typical citrus canker lesions because the lesions were smaller and did not have erumpent margins. Typical yellow Xanthomonas colonies were isolated from the lesions on nutrient agar. The isolates were aerobic, gram negative, rod shaped, and they produced a dark pigment, which is characteristic of some Xanthomonas fuscans subsp. aurantifolii strains. Two reference strains were tested for pathogenicity on not fully expanded leaves of sweet orange, Swingle citrumelo, and key/Mexican lime (C. aurantifolia) plants by wound inoculation with a sterile needle previously dipped in a bacterial suspension (approximately 106 ml-1). Two plants of each species were used for inoculations in greenhouse conditions and six leaves were inoculated per plant. Each inoculated leaf received six point inoculations. These tests confirmed that the host range of this pathogen was restricted to Swingle citrumelo. Symptoms similar to those in the orchard were observed 3 weeks after inoculation and Koch's postulates were completed by reisolation of the bacterium and comparing it with the original isolates. Molecular fingerprinting with PCR-restriction fragment length polymorphism (RFLP) of the 16S-23S spacer region polymorphism (1) and ERIC- and BOX-PCR (2) was used to compare the new strain with 26 reference strains of Xanthomonas citri subsp. citri types A, A* and Aw, X. fuscans subsp. aurantifolii types B and C, and X. alfalfae subsp. citrumelonis. PCR-RFLP and ERIC-PCR showed that this new pathogen had the same profile as X. fuscans subsp. aurantifolii (B and C types). In BOX-PCR, this new strain had a unique profile, but it was still most similar to X. fuscans subsp. aurantifolii and very distinct from X. citri subsp. citri (A, A*, and Aw) and X. alfalfae subsp. citrumelonis strains. During the rainy season in Brazil, this new Xanthomonas strain is less aggressive than X. citri subsp. citri on Swingle citrumelo, inducing fewer lesions without erumpent margins even in young leaves severely infested by the citrus leafminer. The disease only occurred on trees that were separated from each other by 3 to 20 m, suggesting that the bacterium is spread by windblown rain and/or cultural practices. Xanthomonads pathogenic to citrus are of great importance for regulatory purposes worldwide. X. fuscans subsp. aurantifolii is only known to be pathogenic on lemons and limes in the field, and until now, has only been reported to infect lemons and limes in Argentina and key/Mexican lime in São Paulo (Brazil) (3). To our knowledge, this is the first report of a strain of this subspecies that infects Swingle citrumelo but not key/Mexican lime.

First Report of a Group 16SrIII-B Phytoplasma Associated with Decline of China Tree in Brazil.

China tree (Melia azedarach L.), originally from Asia, is an exotic deciduous species in Brazil and is used as an ornamental shade tree in the southern region of the country. Since 2005, plants displaying yellowing, little leaves, witches' broom, and decline have been observed in the State of Rio Grande do Sul. In the streets and avenues of the capital city of Porto Alegre, there are approximately 173 tree species and China tree (6.57% of all trees) is among the top 10 (80,000 China trees and most are symptomatic). Plants with those symptoms are very distinctive and have been found also in the cities of Livramento, Rio Grande, Santa Maria, and Vacaria, places located in seashore areas, and along highways everywhere in the state. The high incidence seems to be related to drought during the last few years. These symptoms are typical of a disease identified by yellowing or decline of China tree associated with phytoplasma and previously reported in the neighboring countries of Argentina, Paraguay, and Bolivia (2). To demonstrate the presence of phytoplasma in diseased trees and to confirm its identity, total DNA was extracted from China tree leaf midribs collected from 10 symptomatic and three asymptomatic plants. Nested PCR was performed with the P1/P7 primer pair in the primary PCR to amplify a 1.8-kb fragment encompassing the 16S rRNA gene, the 16S-23S spacer region, and the 5' end of the 23S rRNA gene, while the secondary PCR was primed by the R16F2n/R16R2 primer pair to amplify a 1.2-kb fragment of the 16S rRNA gene from the 1.8-kb fragment (3,4). DNA fragments of 1.2 kb amplified from nested PCR were analyzed by restriction fragment length polymorphism with restriction enzymes AluI, HhaI, HpaII, KpnI, MboI, MseI, and RsaI, revealing identical profiles for each amplicon and demonstrating that a phytoplasma belonging to group 16SrIII, subgroup B (16SrIII-B) (1) was associated consistently with all symptomatic plants. BLAST analysis revealed 99% identity among these cloned 1.2-kb sequences and representative sequences of phytoplasmas affiliated with group 16SrIII (GenBank Accession Nos. AY081817 and AF147706). A majority consensus sequence representing the phytoplasma found in China trees was selected and deposited in GenBank (Accession No. FJ404775). These results were confirmed by observation with transmission electron microscopy of pleomorphic bodies 400 to 2,000 nm in diameter in the phloem sieve tubes of all symptomatic trees. No phytoplasma was detected or visualized in asymptomatic samples. These results corroborate those from studies conducted in neighboring countries that demonstrated the association between phytoplasmas of group 16SrIII and decline of China trees (1). In conclusion, the current study revealed that a phytoplasma affiliated with group 16SrIII-B is associated with the decline of China tree in Brazil, a disease previously described based solely on symptoms (2). The incidence and severity of the disease are enough to prevent further use of these trees as landscape plants in southern Brazil.

Exploring the Diversity of the Bifidobacterial Population in the Human Intestinal Tract

Although the health-promoting roles of bifidobacteria are widely accepted, the diversity of bifidobacteria among the human intestinal microbiota is still poorly understood. We performed a census of bifidobacterial populations from human intestinal mucosal and fecal samples by plating them on selective medium, coupled with molecular analysis of selected rRNA gene sequences (16S rRNA gene and internally transcribed spacer [ITS] 16S-23S spacer sequences) of isolated colonies. A total of 900 isolates were collected, of which 704 were shown to belong to bifidobacteria. Analyses showed that the culturable bifidobacterial population from intestinal and fecal samples include six main phylogenetic taxa, i.e., Bifidobacterium longum, Bifidobacterium pseudocatenulatum, Bifidobacterium adolescentis, Bifidobacterium pseudolongum, Bifidobacterium breve, and Bifidobacterium bifidum, and two species mostly detected in fecal samples, i.e., Bifidobacterium dentium and Bifidobacterium animalis subp. lactis. Analysis of bifidobacterial distribution based on age of the subject revealed that certain identified bifidobacterial species were exclusively present in the adult human gut microbiota whereas others were found to be widely distributed. We encountered significant intersubject variability and composition differences between fecal and mucosa-adherent bifidobacterial communities. In contrast, a modest diversification of bifidobacterial populations was noticed between different intestinal regions within the same individual (intrasubject variability). Notably, a small number of bifidobacterial isolates were shown to display a wide ecological distribution, thus suggesting that they possess a broad colonization capacity.

Crestamento foliar, nova sintomatologia em algodoeiro causada por Xanthomonas axonopodis pv. malvacearum

Foi observada recentemente no Estado de São Paulo uma nova sintomatologia em algodoeiro cv. Makina, IAC 24 e Detaopal, causada por Xanthomonas axonopodis pv. malvacearum (Xam), denominada "crestamento foliar". Caracteriza-se por crestamento foliar, geralmente acompanhado por halo clorótico, podendo também causar sintomas de "V" invertido, a partir dos bordos foliares. Linhagens de Xam foram comparadas, por meio de testes de patogenicidade, bioquímicos, sorológicos, culturais e PCR-RFLP da região espaçadora 16S-23S DNAr. Independentemente do tipo de sintoma, as linhagens apresentaram características e perfis idênticos aos apresentados pela linhagem tipo, confirmando a identidade dos isolados como Xanthomonas axonopodis pv. malvacearum.

Candidatus Phytoplasma trifolii Associated with Witches' broom of Lespedeza cyrtobotrya M.

The Symptoms of witches` broom disease caused by phytoplasma including general stunting and yellowing, were observed in leafy lespedeza (Lespedeza cyrtobotrya M.) on Doam-myeon, Pyeongchang-gun, in 2006. Based on the sequence analysis of PCR-amplified 16S ribosomal DNA and 16S-23S spacer region DNA products using universal phytoplasma primers, the phytoplasma associated with leafy lespedeza witches` broom (LLWB) disease was identified as a member of Candidatus Pytoplasma trifolii. It was most closely related to alsike clover proliferation phytoplasma (99.8% similarity, accession no. AY390261), Candidatus Pytoplasma trifolii strain. RFLP patterns generated with AluI, HpaII clearly differentiated LLWB phytoplasma from the referenced phytoplasma strains, water dropwort witches` broom, mulberry dwarf, glehni aster yellow dwarf and jujube witches` broom. This paper is the first report on Candidatus Phytoplasma trifolii in leafy lespedeza identified at a molecular level.

Absence of chlamydial infection in Japanese patients with orbital adnexal MALT lymphoma

18523 Background: The extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma) is a distinct B-cell lymphoma that develops in extranodal sites and usually has an indolent clinical course as a localized disease. Recent clinicopathological studies suggest a strong relation between MALT lymphoma and inflammatory diseases of the epithelium such as autoimmune diseases and infections (Sjogren’s syndrome, Hashimoto’s thyroiditis, Helicobacter pylori infection, and etc.), because chronic antigen stimulation has been causally linked to the development of B-cells lymphoproliferative diseases. MALT lymphoma grows in extranodal lymphoid tissue and exists in epithelium tissues, and it is often difficult to distinguish malignant lymphoid tissue from benign inflammation. This is one cause since recent positive or negative correlations between ocular adnexal MALT lymphomas and chlamydial infections have been reported from different researchers, indicate several possible explanations. Methods: Twenty-three cases of ocular adnexal MALT lymphoma in southern regions of Japan, whose monoclonality was confirmed by immunoglobulin heavy chain gene rearrangement and/or cell surface antigens, were analyzed for evidence of several causative factors using ELISA analysis to detect antibodies and Southern blot analysis followed by specific PCR gene amplification. Results: Our series of patients did not show any serological relations to Epstein-Barr virus, Hepatitis C virus, and Chlamydophila psittaci. Two cases had positive serum antibodies for autoimmunity, and another two cases had positive antibodies against Chlamydia trachomatis. None of our cases showed the presence of chlamydial 16S rRNA and 16S-23S spacer rRNA genes (C. psittac, C trachomatis, C. pneumoniae, and C. felis) after Southern blot analysis followed by PCR amplification. Conclusions: Inflammatory agents are still unknown in our series of orbital adnexal MALT lymphomas, and some types of chlamydial infections are not associated with orbital adnexal MALT lymphoma in southern regions of Japan. No significant financial relationships to disclose.

Corynebacterium hansenii sp. nov., an α-glucosidase-negative bacterium related to Corynebacterium xerosis

A novel strain, C-138(T), belonging to the genus Corynebacterium was isolated from a severe thigh liposarcoma infection and its differentiation from Corynebacterium xerosis and Corynebacterium freneyi is described. Analysis of 16S rRNA gene sequences, rpoB sequences and the PCR profile of the 16S-23S spacer regions was not conclusive enough to differentiate strain C-138(T) from C. xerosis and C. freneyi. However, according to DNA-DNA hybridization data, strain C-138(T) constitutes a member of a distinct novel species. It can be differentiated from strains of C. xerosis and C. freneyi by colony morphology, the absence of alpha-glucosidase and some biochemical characteristics such as glucose fermentation at 42 degrees C and carbon assimilation substrates. The name Corynebacterium hansenii sp. nov. is proposed for this novel species; the type strain is C-138(T) (=CIP 108444(T)=CCUG 53252(T)).

Absence of Chlamydial Infection in Japanese Patients with Ocular Adnexal Lymphoma of Mucosa-Associated Lymphoid Tissue

Ocular adnexal extranodal marginal zone B-cell lymphomas of mucosa-associated lymphoid tissue (ocular adnexal MALT lymphoma) are predominately low-grade, small B-cell types and may be caused by several putative inflammatory agents. Twenty-three ocular adnexal MALT lymphoma cases, the monoclonality of which was confirmed by examination of immunoglobulin heavy chain gene rearrangement and/or cell surface antigens, were analyzed for evidence of several causative factors. A serologic evaluation of our series of patients showed no evidence of infection by Epstein-Barr virus, hepatitis C virus, or Chlamydophila psittaci. Two patients tested positive for serum antibodies for autoimmunity, and another 2 patients tested positive for antibodies against Chlamydia trachomatis. Polymerase chain reaction analysis did not reveal the presence of the chlamydial 16S ribosomal RNA (rRNA) gene or the 16S-23S spacer rRNA gene. These results indicate that the inflammatory agents in our series of ocular adnexal MALT lymphomas are still unknown and that some types of chlamydial infections are not associated with orbital adnexal MALT lymphoma in southern regions of Japan.

Detection, Identification and Molecular Characterization of a Phytoplasma Associated with Arabian Jasmine (Jasminum sambac L.) Witches’ Broom in Oman

AbstractArabian jasmine (Jasminum sambac L.) plants showing witches’ broom (WB) symptoms were found in two regions in the Sultanate of Oman. Polymerase chain reaction (PCR) amplification of the 16S rRNA gene and the 16S–23S spacer region utilizing phytoplasma‐specific universal and designed primer pairs, and transmission electron microscopy of phytoplasma‐like structures in phloem elements confirmed phytoplasma infection in the symptomatic plants. PCR products primed with the P1/P7 primer pair were 1804 bp for jasmine witches’ broom (JasWB) and 1805 bp for alfalfa (Medicago sativa L.) witches’ broom (AlfWB). Actual and putative restriction fragment length polymorphic analysis indicated that jasmine and AlfWB phytoplasmas were molecularly indistinguishable from each other and closely related to papaya yellow crinkle (PYC), as well as being distinct from lime WB (LWB) and Omani alfalfa WB (OmAlfWB) phytoplasmas. A sequence homology search of JasWB and AlfWB showed 99.8% similarity with PYC from New Zealand and 99.6% similarity with each other (JasWB/AlfWB). The jasmine and AlfWB phytoplasmas were also shown to be related to the peanut WB group (16SrII) of 16S rRNA groups based on a phylogenetic tree generated from phytoplasma strains primed with the P1/P7 primer pair and representing the 15 phytoplasma groups.

DIFFERENTIAL AMPLIFICATION OF ACHOLEPLASMA LAIDLAWII PG8 rrnA AND rrnB NUCLEOTIDE SEQUENCES DURING DISSOCIATION OF THE CELL CULTURE POPULATION

ABSTRACT The amplification of the 16S–23S spacer areas of the Acholeplasma laidlawii PG8 rRNA rrnA and rrnB operons has its own features. The attenuation of the polymerase chain reaction (PCR) signal of the nucleotide sequences of rrnA containing tRNA genes might be observed if DNA without enzyme deproteinization is used as a matrix. The phenomenon takes place due to dissociation of cell population caused by the active entering of the vegetative cell forms into ultramicroforms in the mycoplasma culture at unfavorable growth conditions. The DNA of A. laidlawii PG8 ultramicroforms showed selective amplification of the rrnB nucleotide sequences – tRNA‐free rRNA operon. As to vegetative cells, the “equal” PCR signals for the nucleotide of rrnA and rrnB sequences were registered. In this connection, the use of specific nucleotide sequences of the rrnA spacer area as primers for PCR, as well as the mycoplasma cell DNA without special enzyme deproteinization as a matrix, may lead to wrong conclusions about the presence of A. laidlawii in the tested samples. The ability of A. laidlawii PG8 vegetative cell forms of actively entering into ultramicroforms at unfavorable growth conditions seems to demand a new approach to control mycoplasma infections, providing an efficient diagnosis to detect the vegetative cell forms and ultramicroforms of the mycoplasma in the tested samples.

Absence of Chlamydial Infection in Japanese Patients with Ocular Adnexal MALT Lymphoma.

The extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma) is a distinct type of B-cell lymphoma that develops in extranodal sites and usually has an indolent clinical course as a localized disease. Recent clinicopathological studies suggest a strong relationship between MALT lymphoma and inflammatory diseases of the epithelium such as autoimmune diseases and infections (Sjogren's syndrome, Hashimoto's thyroiditis, Helicobacter pylori infection, and etc.), because chronic antigen stimulation has been causally linked to the development of lymphoproliferative diseases in B cells. MALT lymphoma grows in extranodal lymphoid tissue and exists in epithelium tissues, and it is often difficult to distinguish malignant lymphoid tissue from benign inflammation. However, this is only one of the possible causes; recent positive and negative correlations between ocular adnexal MALT lymphomas and chlamydial infections reported by different researchers indicate several different possible explanations. Here, we analyze twenty-three cases of ocular adnexal MALT lymphoma whose monoclonality was confirmed by immunoglobulin heavy chain gene rearrangement and/or the analysis of cell surface antigens for evidence of expected causative factors, using both ELISA analysis to detect antibodies and specific PCR gene amplification followed by Southern blot analysis. Our series of patients did not show any serological relations to the Epstein-Barr virus, the Hepatitis C virus, or to Chlamydophila psittaci. Two cases had positive serum antibodies for autoimmunity, another two cases had positive antibodies against Chlamydia trachomatis. None of our cases showed the presence of chlamydial 16S rRNA or 16S-23S spacer rRNA genes (C. psittac, C trachomatis, C. pneumoniae, and C. felis) after PCR amplification. These results indicate that the inflammatory agents are still unknown in our series of ocular adnexal MALT lymphomas, and that some types of chlamydial infections are not associated with ocular adnexal MALT lymphoma in southern regions of Japan.

Identification of cyanobacteria by polymorphisms of PCR-amplified ribosomal DNA spacer region

Abstract The 16S-23S ribosomal DNA spacer region of selected cyanobacterial strains was amplified by the polymerase chain reaction using primers to conserved flanking sequences. Single or multiple rDNA amplification products were generated depending on the strain and primer pair. Species could generally be distinguished on the basis of size heterogeneity of the products. Analysis of restriction digests of the amplified rDNAs indicated polymorphisms useful in identification. Four enzymes ( Hin fI, Dde I, Alu I, Taq I) generated restriction fragment length patterns that could discriminate between the cyanobacteria to the taxonomic levels of genus and species. This approach should prove useful in the rapid identification of cyanobacteria.