16S-23S Intergenic Spacer Region Research Articles

Comparative Analysis of Tick-Borne Relapsing Fever Spirochaetes from Ethiopia and Nigeria.

Despite increasing reports of tick-borne diseases in Africa, remarkably, reports of tick-borne relapsing fever (TBRF) in Nigeria are lacking. Ornithodoros savignyi from Nigeria have been reported with the relapsing fever Candidatus Borrelia kalaharica. Conversely, in Ethiopia, the agent of relapsing fever is the louse-borne relapsing fever (LBRF) spirochaete Borrelia recurrentis with no TBRF reported to occur. A total of 389 Ornithodoros ticks, Ethiopia (N = 312) and Nigeria (N = 77), were sampled, together with 350 cattle, and 200 goat sera were collected from Nigeria. Samples were screened for Borrelia spp. by RT-PCR. Reactive samples were confirmed, then sequenced using flagellin B, 16S rRNA, and 16S-23S intergenic spacer region. The prevalence of Borrelia spp. in livestock was 3.8% (21/550) and 14% (3/21) after final molecular confirmation. Of 312 ticks from Ethiopia, 3.5% (11/312) were positive for Borrelia, with 36% (4/11) by conventional PCR. Sequencing revealed that the borreliae in soft ticks was C. B. kalaharica, whilst that found in animals was Borrelia theileri. Soft ticks were confirmed by sequencing 7% (22/312) and 12% (9/77) of the Ethiopian and Nigerian ticks, respectively. Phylogenetic analysis revealed that these were Ornithodoros savignyi. This is the first evidence of C. B. kalaharica in Ethiopia and demonstrates the co-existence of TBRF in a country endemic to LBRF. Important, this might cause a diagnostic challenge given that LBRF is predominantly diagnosed by microscopy, which cannot differentiate these two spirochaetes. Furthermore, we report B. theileri in ruminants in Nigeria, which may also be of veterinary and economic importance.

Multilocus Sequence Analysis Reveals New Hosts of Palm Lethal Decline Phytoplasmas in Florida, U.S.A.

Palm-infecting phytoplasmas have been a persistent threat to sustainable palm production in Florida, U.S.A., for nearly a century. First, lethal yellowing (LY) was introduced in the early 1900s, then lethal bronzing (LB) was introduced around 2006. While LY is currently rare in Florida, LB is actively spreading throughout the state and as it becomes more established, has begun infecting new host species. Continual monitoring efforts throughout the state seeks to catch infections and remove palms before further spread. As a result of this effort, many palms displaying a variety of symptoms are sampled and submitted to the University of Florida – Fort Lauderdale Research and Education Center (FLREC) for confirmation of phytoplasma infection status. Over the course of 3 years, multiple samples of Washingtonia robusta and Roystonea regia were submitted. All of these samples were negative for phytoplasma upon the initial qPCR screening and were tested with universal primers and confirmed as negative. Specimens of Arenga engleri, Rhapidophyllum hystrix, and Sabal palmetto were received and tested positive for LB, LB, and LY, respectively, tentatively representing new host records for these species and respective phytoplasma. Multilocus sequence analyses utilizing the 16S rRNA, 16S-23S intergenic spacer region, secA, secY, and groEL further confirmed the identity of LB and LY in the respective new hosts. These results highlight the need for continued monitoring of palms in Florida so that infected palms can be removed quickly. Also, the novel molecular data ( secY) help better understand and identify phytoplasmas in palms.

Mycoplasma tauri sp. nov. isolated from the bovine genital tract

Since 2006, a Mycoplasma species unidentifiable to the species level has been regularly isolated from the semen and prepuce of apparently healthy bulls, and occasionally from cattle displaying inflammatory disease of the genital tract. Seven of these Mycoplasma isolates were subjected to a comprehensive taxonomic study. The strains investigated grew well in modified Hayflick’s medium and colonies on agar exhibited typical fried egg morphology and produced ‘film and spots’. Transmission electron microscopy revealed a cell morphology characteristic of mycoplasmas with spherically shaped cells bounded by a bi-layered cell membrane. The strains studied neither produced acid from sugar carbon sources nor did hydrolyse arginine or urea, and genome annotation indicated that organic acids (pyruvate, lactate) are used as energy sources. Phylogenetic analyses of 16S rRNA gene sequences, the 16S-23S intergenic spacer region, and partial rpoB gene and protein sequences placed the strains within the Mycoplasma (M.) bovis cluster of the Hominis group with M. primatum, M. agalactiae, and M. bovis being their closest relatives. Genomic information including whole-genome similarity metrics (ANIb, ANIm, TETRA, dDDH, AAI) and phylogenomics, proteomic features revealed by matrix-assisted laser desorption ionization time of flight (MALDI-ToF) mass spectrometry as well as serological reactions and polar lipid profiling strongly indicated that the strains examined were representatives of a hitherto unclassified species of genus Mycoplasma, for which the name Mycoplasma tauri sp. nov. with type strain Zaradi2T (=ATCC BAA-1891T = DSM 22451T) is proposed.

Genetic Variability of Palm Lethal Decline Phytoplasmas in the Caribbean Basin and Florida, U.S.A., Based on a Multilocus Analysis.

Palm lethal decline phytoplasmas are an important group of plant pathogens that cause death in a variety of palm species throughout the Caribbean basin and the southeastern United States. The 16SrIV-D phytoplasma was introduced to the state of Florida, United States; it has since caused severe economic losses to the green industries of Florida and is threating natural ecosystems because of its ability to infect the native palm Sabal palmetto. In this study, the genetic variability of the 16SrIV-D phytoplasma was assessed over a 10-year period to determine if multiple introductions had occurred or if natural mutations were occurring. Furthermore, the genetic variability of the palm lethal decline phytoplasma group (16SrIV) was assessed with a multiple locus analysis using the 16S ribosomal RNA gene, the 16S-23S intergenic spacer region, and secA and groEL genes. Overall, no variability of the 16SrIV-D phytoplasma was documented in Florida over a 10-year period. The multilocus analysis showed support for three distinct species of the phytoplasma in the Caribbean basin that infect palms and further support that the 16SrIV-C from Tanzania is not closely related. Furthermore, 16SrIV-B and 16SrIV-D were found to be the same phytoplasma based on 100% identity between the two based on intergenic spacer region, secA, and groEL analysis. This study represents the first robust, multilocus analysis of palm-infecting phytoplasmas from the Caribbean and sheds light on the phylogeny and evolution of the group.

Characterization of streptococcus-like bacteria from diseased Oreochromis niloticus farmed on Lake Kariba in Zambia

Tilapia (Oreochromis niloticus) farming on Lake Kariba, Zambia has been practiced since early 2000s. In recent years, the industry has experienced disease outbreaks with clinical signs consistent with streptococcus-like infections. Affected fish were growers weighing approximately 200 g or above from farms designated A and B, reared in cages on the lake. The objective of this study was to identify and characterize the causative bacteria of these outbreaks. A total of eighty-six diseased fish were sampled between 2014 and 2016. Bacteria were cultured from internal organs and were characterized by biochemical tests, PCR and sequencing of the 16S rRNA gene or 16S23S intergenic spacer region. The results showed the prevalence of Lactococcus garvieae at farm A in 2015 and 2016 outbreaks (15/20 and 23/40, respectively) and at farm B (6/20) in 2016. Infection of naïve fish with one isolate of L. garvieae recovered from the outbreaks resulted in clinical signs and mortalities akin of what had been observed during the outbreaks, confirming Koch's postulates. In the 2014 outbreaks, the low prevalence of Streptococcus agalactiae (4/15 and 2/11 respectively at farms A and B) and S. iniae (2/15) at farm A suggests that other pathogens were likely involved in the disease outbreak observed.

Genetic diversity of Streptococcus equi subsp. zooepidemicus isolated from horses

Streptococcus equi subsp. zooepidemicus (SEZ) is an opportunistic and zoonotic pathogen of horses. In this study, genetic intraspecies variability of SEZ obtained mainly from respiratory and genital samples of horses was investigated by analysis of the 16S–23S rRNA intergenic spacer region (ISR) and of the 16S rRNA gene. 16S–23S ISR rRNA type A1 was predominant, although a high rate of multiple products (30.5%) was obtained. Phylogenetic analysis of the 16S rRNA gene detected three genogroups (I, II and III). 16S rRNA variable regions V1 and V2 are the most important regions for evaluating SEZ intraspecies variability, but at least V1-V5 regions should be considered to avoid mistakes. Analysis of all 16S rRNA sequences available in databases assigned human SEZ to groups I and III but not to group II. These results show a high genetic variability in SEZ collected from different specimens of horses from various regions of Italy.

Prevalence and Phylogenetic Analysis of Mycoplasma Synoviae Strains Isolated from Polish Chicken Layer Flocks.

IntroductionMycoplasma synoviae (MS) is a chicken pathogen of major economic importance.Material and MethodsBetween 2010 and 2016, 906 commercial layer chicken flocks in Poland were examined for MS, and the phylogenetic relationship among the strains was established. Regionally dispersed samples were collected and tested with the use of real-time PCR to detect the 16S–23S intergenic spacer region. Positive samples were also tested with LAMP and conventional PCR to detect the vlhA gene.ResultsMS genetic material was detected in 265 (29%) of the tested flocks by real-time PCR, in 227 by the LAMP method and in 202 (22%) by conventional PCR. The by-year percentage of positive samples began at 34% in 2010, rose to 44% in 2012, and declined to 29% in 2016. A phylogenetic analysis of Polish M. synoviae strains using a partial sequence of the vlhA gene showed nine genotypes (A–I), the most frequently occurring being F and C. Pathogenic Polish MS field isolates (n = 27) collected from chickens with clinical signs of infection were grouped for their characteristic symptoms: respiratory for genotypes C, E, F, and I (n = 13), EAA and a drop in laying for genotypes F, E, and C (n = 12), and synovitis for genotype A (n = 2).ConclusionThese data showed the country’s isolate diversity. The high prevalence suggests the need to introduce appropriate control programmes. This is the first report of molecular epidemiological data on M. synoviae infection in layer chickens in Poland.

Characterization and Differentiation of Chicken Mycoplasma Isolates Using 16S-23S Intergenic Spacer Region Sequencing

ABSTRACT The objective of this study was to identify the species and characterize the genetic relationships among mycoplasma isolates from commercial layer hen flocks using 16S-23S rDNA intergenic spacer region (IGSR) sequencing. Twenty-one isolates were obtained from samples collected from commercial layer flocks in four Brazilian states: Sao Paulo, Minas Gerais, Rio de Janeiro and Espirito Santo. The isolates were recovered from the Sao Paulo, Rio de Janeiro and Espirito Santo states. Eleven isolates were originated from tracheal swabs, five from shell gland swabs and five from ovary fragment collection. The 16S-23S rDNA IGSR of isolates were amplified by PCR, and the obtained products were subsequently sequenced. The consensus of each isolate was compared to the available sequences using Nucleotide BLAST® to determine the mycoplasma species. A phylogenetic analysis of the Mycoplasma gallisepticum (MG) sequences was performed. Pairwise analyses showed homologies of 99% to 100% with the previously characterized sequences listed in GenBank®. Four Mycoplasma gallinaceum were isolated from three flocks and seven M. pullorum isolates were obtained from a single flock. The other 10 isolates were all identified as MG and were obtained from four flocks. The 16S-23S rDNA IGSR sequencing was a good method to identify Mycoplasma species isolated from field samples, providing fast and reliable results at relatively low costs. The results were also satisfactory for the single-locus sequence typing of MG isolates.

Mycoplasmosis of House Finches ( Haemorhous mexicanus) and California Scrub-Jays ( Aphelocoma californica) in a Wildlife Rehabilitation Facility with Probable Nosocomial Transmission.

We describe an investigation of an outbreak of conjunctivitis in juvenile House Finches ( Haemorhous mexicanus) and California Scrub-jays ( Aphelocoma californica) at a central California, US wildlife rehabilitation facility. In late May 2015, the facility began admitting juvenile finches, the majority with normal eyes at intake. In June, with juvenile finches already present, the facility admitted juvenile scrub-jays, all with normal eyes at intake. In July, after conjunctivitis was observed in increasing numbers of juvenile finches and scrub-jays, carcasses were submitted for postmortem examination. Histopathology of five finches and three scrub-jays identified lymphocytic infiltrates in the ocular tissues. Conjunctival swabs from 87% (13/15) finches and 33% (4/12) scrub-jays were PCR-positive for Mycoplasma gallisepticum. One finch and two scrub-jays were PCR-positive for Mycoplasma synoviae. Additionally, gene sequencing (16S ribosomal RNA and 16S-23S intergenic spacer region) identified Mycoplasma sturni from 33% (3/9) scrub-jays. This outbreak of conjunctivitis suggested that M. gallisepticum-infected juvenile finches admitted to and maintained in a multispecies nursery likely resulted in transmission within the facility to healthy juvenile finches and scrub-jays. Evidence of other Mycoplasma spp. in finches and scrub-jays indicates that these species are susceptible to infection and may act as carriers. This outbreak highlighted the need for effective triage and biosecurity measures within wildlife rehabilitation facilities.

Technological properties assessment and two component systems distribution of Streptococcus thermophilus strains isolated from fermented milk.

Streptococcus thermophilus is one of the economically most representatives of lactic acid bacteria, which is widely used as a starter to produce fermented milk products. In this study, 22 S. thermophilus strains were isolated from 26 fermented milk samples. Most isolates showed the ability to ferment a broad range of carbohydrates. Interestingly, eight strains are galactose positive, which is a desirable property in various industrial dairy fermentations. Four different nucleotide sequences were found in the galR-galK intergenic regions. The 16S-23S intergenic spacer region sequences of most isolates were determined as ITS-St-II type, which are related with protease positive and fast acidification. CS18 presented excellent technological performances, and showed potential as a promising starter candidate. To gain a comprehensive view of stress response mechanisms of strains, the distribution of all the two-component systems (TCSs) in strains were investigated. TCS analysis indicated that the nucleotide sequence of TCSs have obvious differences in different strains. And the strains with the special nucleotide sequences of TCS have distinctive traits. Therefore, it was speculated that there is a certain connection between the traits' difference and the TCS difference of strains.

Rapid Acquisition of Linezolid Resistance in Methicillin-Resistant Staphylococcus aureus: Role of Hypermutation and Homologous Recombination.

BackgroundWe previously reported the case of a 64-year-old man with mediastinitis caused by Staphylococcus aureus in which the infecting bacterium acquired linezolid resistance after only 14 days treatment with linezolid. We therefore investigated relevant clinical isolates for possible mechanisms of this rapid acquisition of linezolid resistance.MethodsUsing clinical S. aureus isolates, we assessed the in vitro mutation rate and performed stepwise selection for linezolid resistance. To investigate homologous recombination, sequences were determined for each of the 23S ribosomal RNA (23S rRNA) loci; analyzed sequences spanned the entirety of each 23S rRNA gene, including domain V, as well as the 16S-23S intergenic spacer regions. We additionally performed next-generation sequencing on clinical strains to identify single-nucleotide polymorphisms compared to the N315 genome.ResultsStrains isolated from the patient prior to linezolid exposure (M5-M7) showed higher-level linezolid resistance than N315, and the pre-exposure strain (M2) exhibited more rapid acquisition of linezolid resistance than did N315. However, the mutation rates of these and contemporaneous clinical isolates were similar to those of N315, and the isolates did not exhibit any mutations in hypermutation-related genes. Sequences of the 23S rRNA genes and 16S-23S intergenic spacer regions were identical among the pre- and post-exposure clinical strains. Notably, all of the pre-exposure isolates harbored a recQ missense mutation (Glu69Asp) with respect to N315; such a lesion may have affected short sequence recombination (facilitating, for example, recombination among rrn loci). We hypothesize that this mechanism contributed to rapid acquisition of linezolid resistance.ConclusionsHypermutation and homologous recombination of the ribosomal RNA genes, including 23S rRNA genes, appear not to have been sources of the accelerated acquisition of linezolid resistance observed in our clinical case. Increased frequency of short sequence recombination may have resulted from a recQ variant in the infecting organism.

Fleas and Flea-AssociatedBartonellaSpecies in Dogs and Cats from Peru: Table 1.

In the present study, we investigated 238 fleas collected from cats and dogs in three regions of Peru (Ancash, Cajamarca, and Lima) for the presence of Bartonella DNA. Bartonella spp. were detected by amplification of the citrate synthase gene (16.4%) and the 16S-23S intergenic spacer region (20.6%). Bartonella rochalimae was the most common species detected followed by Bartonella clarridgeiae and Bartonella henselae. Our results demonstrate that dogs and cats in Peru are infested with fleas harboring zoonotic Bartonella spp. and these infected fleas could pose a disease risk for humans.

First report of a 16SrVI group related phytoplasma associated with cucumber phyllody in a greenhouse in Iran

Greenhouse cucumber (Cucumis sativus) is one of the most economically important vegetable crops in Iran. In September 2014, an outbreak of phyllody disease was observed in up to 82% of greenhouse cucumber in cultivars Keyhan and Negin in Nasr Abad (Taft, Yazd province). Symptoms were characterised by flower virescence, phyllody and sterility (Fig.1). Total DNA was extracted from 0.2 g of fresh phyllody-affected flowers and from symptomless cucumber plants (Zhang et al., 5). DNA samples were tested for phytoplasma by direct and nested PCR using the phytoplasma primers P1/P7 (Deng & Hiruki, 2; Schneider et al., 4) followed by R16F2n/R16R2 (Gundersen & Lee, 3). Primer pair P1/P7 amplifies an 1800 bp fragment of the phytoplasma ribosomal operon which includes the 16SrRNA gene, the 16S-23S intergenic spacer region (SR) and a portion of the 5' region of the 23S rRNA gene. R16F2n/R16R2 primer pair amplifies about 1250 bp of the phytoplasma 16S rRNA gene. PCR amplicons of ~1.8 and ~1.25 kbp were obtained from symptom-bearing, but not from symptomless greenhouse cucumber plants. Seven R16F2nR16R2 amplicons from infected areas in Nasr Abad were directly sequenced. The R16F2n/R16R2 sequences shared 100% sequence identity. A consensus sequence corresponding to the Saleh Abad (Nasr Abad, Taft, Yazd province) greenhouse cucumber phyllody (SGCP) was deposited in GenBank (Accession No. KR633068). BLAST search showed that the SGCP phytoplasma had the highest sequence identity with those of the 16SrVI (‘Candidatus Phytoplasma trifolii’) phytoplasma group. Phylogenetic analysis (MEGA software version 6.0) confirmed that the SGCP phytoplasma clustered within the 16SrVI phytoplasma clade (Fig. 2). R16F2n/R16R2 SGCP phytoplasma sequence computer-simulated restriction analysis (Fig.3) with restriction endonucleases AluI, BamHI, BfaI, BstUI, DraI, EcoRI, HaeIII, HhaI, HinfI, HpaI, HpaII, KpnI, Sau3AI, MseI, RsaI, SspI and TaqI using iPhyclassifier (Zhao et. al., 6) revealed that virtual RFLP patterns of the SGCP phytoplasma differ from those previously recorded 16SrVI subgroups (A, B, C, D, E, F, H, I). Although the SGCP phytoplasma RFLP profiles were more similar to those of subgroup A (AY390261), it showed a similarity coefficient of 0.93; which suggests that the SGCP phytoplasma may represent a new subgroup within the 16SrVI phytoplasma group. This is the first report of the association of a 16SrVI phytoplasma with cucumber phyllody disease in a greenhouse in Iran. A 16SrII phytoplasma (‘Ca. P. aurantifolia’) was associated with cucumber phyllody in a greenhouse of the Fars province (Dehghan et al., 1). Therefore the SGCP phytoplasma may represent a new epidemiological constraint for the development of the phyllody disease in greenhouses in Iran.

First report of a 16SrI‐B subgroup‐related phytoplasma associated with Eruca sativa phyllody in Iran

During a 2012-14 survey for phytoplasma diseases, Eruca sativa phyllody (ESP) disease was observed in Abarkooh (Yazd province, Iran). Infected plants showed crown proliferation, witches' broom, little leaf, flower virescence, phyllody, sterility and stunting (Fig. 1). Total DNA was extracted from 0.2 g of fresh leaves and flowers from both infected and symptomless plants (Zhang et al., 5). DNA samples were tested for phytoplasma by direct PCR using primer pair P1/P7 (Deng & Hiruki, 1; Schneider et al., 4) and nested PCR using primer pairs P1/P7 and R16F2n/R16R2 (Gundersen & Lee, 2). Primer pair P1/P7 amplifies an 1.8 kb fragment of the phytoplasma ribosomal RNA operon which includes the 16S rRNA gene, the 16S-23S intergenic spacer region and a portion of the 5‘ region of the 23S rRNA gene. The R16F2n/R16R2 primer pair amplifies approximately 1.25 kb of the phytoplasma 16S rRNA gene. PCR amplicons of ~1.8 and ~1.25 kb respectively were obtained from all 10 of the phyllody-affected E. sativa (two per location) but not from symptomless plants. Five R16F2n/R16R2 amplicons from Esfandabad, Harooni, Bedaf, Taqhiabad and Mehrdasht locations were directly sequenced and showed 100% sequence identity to each other. A consensus sequence corresponding to the Aliabad-e Shams (Esfandabad, Abarkooh, Yazd province) E. sativa phyllody (AESP) phytoplasma was deposited in GenBank (Accession No. KT626568). A BLAST search showed that the AESP phytoplasma had 100% sequence identity with those of the 16SrI ‘Candidatus Phytoplasma asteris' group (Aster Yellows group), including the onion yellows phytoplasma mild strain (OY-M) (NC_005303) representative of the 16SrI-B subgroup. Phylogenetic analysis (MEGA software version 6.0) confirmed that the AESP phytoplasma clustered within the 16SrI phytoplasma clade (Fig. 2) closer to the 16SrI-B strains, OY-M and ‘Ca. P. asteris’ (M30790). Computer-simulated restriction analysis (Fig. 3) with 17 restriction endonucleases using iPhyClassifier (Zhao et al., 6) revealed that virtual RFLP patterns of the AESP phytoplasma were identical (similarity coefficient 1.00) to those of OY-M. This is the first report of a 16SrI-B phytoplasma associated with ESP disease in Iran. A 16SrI-B related phytoplasma has been reported associated with rapeseed (Brassica rapa) phyllody in Iran (Salehi et al., 3). Eruca sativa may be a secondary host for the 16SrI-B phytoplasma associated with rapeseed phyllody.

A newly established real-time PCR for detection of Borrelia miyamotoi in Ixodes ricinus ticks

A total of 350 ticks collected in Austria were analyzed for the presence of DNA sequences of B. miyamotoi. Three ticks gave positive results in a B. miyamotoi-specific nested PCR. Results were confirmed by sequencing the amplified glpQ gene from the positive samples. Moreover we developed a real-time PCR which unambiguously detected B. miyamotoi in all positive samples. Further genotyping of the samples found 100% identity of the 16S–23S intergenic spacer region with Swedish B. miyamotoi sequences. This is the first detection of the relapsing fever spirochete Borrelia miyamotoi in hard ticks in Austria. The results consolidate the picture of a European-wide distribution of B. miyamotoi and again underscore the need for clinical awareness to clarify possible involvement of this species in human disease.

Molecular Analysis of Genetic Diversity of Pectobacterium carotovorum Subsp Carotovorum Isolated in Morocco by PCR Amplification of the 16S-23S Intergenic Spacer Region

Aims: Pectobacterium carotovorum is a ubiquitous bacterium that causes soft rot in different crop plants throughout the world. In Morocco, approximately 95% of the Strains isolates from potato plants with tuber soft rot are P. carotovorum. In this study, we test whether PCR ribotyping can be used to distinguish strains of Pectobacterium carotovorum isolated from soft rot potato and to differentiate among strains from different geographic regions. Place and Duration of Study: Laboratory of Virology, Microbiology and Quality / Ecotoxicology and Biodiversity, department Biology, Faculty of Sciences and Techniques, University Hassan-II Mohammedia Casablanca. Methodology: Eighty-three pectolytic enterobacteria were collected from potatoes rotten in Morocco, the strains were isolated in the Cristal Violet Pectate (CVP) medium and were purified in Original Research Article Amdan et al.; BBJ, 7(3): 102-110, 2015; Article no.BBJ.2015.051 103 LPGA agar (yeast extract, peptone, glucose and agar). After purification, strains were identified by physiological and biochemical tests. The confirmation of species was performed by PCR using primers Y1 and Y2. The genetic diversity of Pectobacterium carotovoum was investigated by PCR ribotyping using primers G1/L1, which are complementary to conserved regions of the rRNA operon. Furthermore, the profiles obtained were compared by the Unweighted Pair Group Method. Results: The biochemical and physiological analysis demonstrated that the predominant pectolytic enterobacterium present in Morocco is Pectobacterium carotovorum subsp carotovorum. The specific confirmation of species P. carotovorum by PCR has yielded a 434 bp DNA fragment of the pelY gene with all isolates. Further, PCR amplification of the 16S-23S Intergenic spacer Region (ITS-PCR) has presented a specific pattern made of 2-6 fragments ranging from 300 bp to 800 bp. The UPGMA tree has shown that there is considerable genetic diversity in P. carotovorum strains, which can be divided into four distinct groups.

A Mycoplasma species of Emydidae turtles in the northeastern USA.

Mycoplasma infections can cause significant morbidity and mortality in captive and wild chelonians. As part of a health assessment of endangered bog turtles (Glyptemys muhlenbergii) in the northeastern US, choanal and cloacal swabs from these and other sympatric species, including spotted turtles (Clemmys guttata), eastern box turtles (Terrapene carolina carolina), wood turtles (Glyptemys insculpta), and common snapping turtles (Chelydra serpentina) from 10 sampling sites in the states (US) of Delaware, New Jersey, and Pennsylvania, were tested by PCR for Mycoplasma. Of 108 turtles tested, 63 (58.3%) were PCR positive for Mycoplasma including 58 of 83 bog turtles (70%), three of three (100%) eastern box turtles, and two of 11 (18%) spotted turtles; all snapping turtles (n = 7) and wood turtles (n = 4) were negative. Sequence analysis of portions of the 16S-23S intergenic spacer region and the 16S ribosomal RNA gene revealed a single, unclassified species of Mycoplasma that has been previously reported in eastern box turtles, ornate box turtles (Terrapene ornata ornata), western pond turtles (Emys marmorata), and red-eared sliders (Trachemys scripta elegans). We document a high incidence of Mycoplasma, in the absence of clinical disease, in wild emydid turtles. These findings, along with wide distribution of the identified Mycoplasma sp. across a broad geographic region, suggest this bacterium is likely a commensal inhabitant of bog turtles, and possibly other species of emydid turtles, in the northeastern US.

First Report of Aster Yellows Phytoplasma (16SrI‐B) Associated with Witches' Broom Disease of Melia azedarach var. japonica in Korea

AbstractMelia azedarach var. japonica trees with leaf yellowing, small leaves and witches' broom were observed for the first time in Korea. A phytoplasma from the symptomatic leaves was identified based on the 16Sr DNA sequence as a member of aster yellows group, ribosomal subgroup 16SrI‐B. Sequence analyses of more variable regions such as 16S–23S intergenic spacer region, secY gene, ribosomal protein (rp) operon and tuf gene showed 99.5−100% nucleotide identity to several GenBank sequences of group 16SrI phytoplasmas. Phylogenetic analysis confirmed that the Melia azedarach witches' broom phytoplasma belongs to aster yellows group.

Phenotypic characterization and genetic diversity of Flavobacterium columnare isolated from red tilapia, Oreochromis sp., in Thailand.

Flavobacterium columnare is the aetiological agent of columnaris disease and severely affects various freshwater aquaculture fish species worldwide. The objectives of this study were to determine the phenotypic characteristics and genetic variability among F.columnare isolates isolated from red tilapia in Thailand. Forty-four F.columnare isolates were recovered from diseased fish in different geographical locations. The isolates exhibited homologous phenotypic characteristics but exhibited genetic diversity. One isolate was assigned to genomovar I, and the remainder were assigned to genomovar II, indicating the coexistence of these genomovars but predominance of genomovar II. Phylogenetic analysis of the 16S-23S ISR sequences revealed that a subset of the Thai isolates (n=25) contained a smaller intergenic spacer region (ISR) (523-537bp) and formed a unique ISR phylogenetic group. Phylogenetic analysis of the 16S rRNA gene supported the unique cluster of Thai isolates. This is the first description of the phenotypic and molecular characteristics of F.columnare isolated from red tilapia in Thailand as well as five isolates of F.columnare derived from other fish species including Nile tilapia, koi carp and striped catfish.

Recombination drives evolution of the Clostridium difficile 16S-23S rRNA intergenic spacer region.

PCR-ribotyping, a typing method based on size variation in 16S-23S rRNA intergenic spacer region (ISR), has been used widely for molecular epidemiological investigations of C. difficile infections. In the present study, we describe the sequence diversity of ISRs from 43 C. difficile strains, representing different PCR-ribotypes and suggest homologous recombination as a possible mechanism driving the evolution of 16S-23S rRNA ISRs. ISRs of 45 different lengths (ranging from 185 bp to 564 bp) were found among 458 ISRs. All ISRs could be described with one of the 22 different structural groups defined by the presence or absence of different sequence modules; tRNAAla genes and different combinations of spacers of different lengths (33 bp, 53 bp or 20 bp) and 9 bp direct repeats separating the spacers. The ISR structural group, in most cases, coincided with the sequence length. ISRs that were of the same lengths had also very similar nucleotide sequence, suggesting that ISRs were not suitable for discriminating between different strains based only on the ISR sequence. Despite large variations in the length, the alignment of ISR sequences, based on the primary sequence and secondary structure information, revealed many conserved regions which were mainly involved in maturation of pre-rRNA. Phylogenetic analysis of the ISR alignment yielded strong evidence for intra- and inter-homologous recombination which could be one of the mechanisms driving the evolution of C. difficile 16S-23S ISRs. The modular structure of the ISR, the high sequence similarities of ISRs of the same sizes and the presence of homologous recombination also suggest that different copies of C. difficile 16S-23S rRNA ISR are evolving in concert.