bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of a Phytoplasma Associated with Frogskin Disease in Cassava
Authors: Alvarez Elizabeth , Mejía Juan F. , Llano Germán A. , Loke John B. , Calari Alberto , Duduk Bojan , Bertaccini Assunta
Published: October 2009

Cassava frogskin disease (CFSD) is an economically important root disease of cassava (Manihot esculenta) in Colombia and other South American countries, including Brazil, Venezuela, Peru, Costa Rica, and Panama. The roots of severely affected plants are thin, making them unsuitable for consumption. In Colombia, phytoplasma infections were confirmed in 35 of 39 genotypes exhibiting mild or severe CFSD symptoms either by direct or nested polymerase chain reaction (PCR) assays employing ribosomal (r)RNA operon primer pairs. The CFSD-associated phytoplasmas were identified as group 16SrIII strains by restriction fragment length polymorphism (RFLP) and sequence analyses of amplified rDNA products, and results were corroborated by PCRs employing group 16SrIII-specific rRNA gene or ribosomal protein (rp) gene primers. Collectively, RFLP analyses indicated that CFSD strains differed from all phytoplasmas described previously in group 16SrIII and, on this basis, the strains were tentatively assigned to new ribosomal and ribosomal protein subgroups 16SrIII-L and rpIII-H, respectively. This is the first molecular identification of a phytoplasma associated with CFSD in cassava in Colombia.
Susceptibility of Populus balsamifera to Septoria musiva: A Field Study and Greenhouse Experiment
Authors: LeBoldus Jared M. , Blenis Peter V. , Thomas Barb R. , Feau Nicolas , Bernier Louis
Published: October 2009

A greenhouse inoculation experiment and field study were conducted to determine the cause of an outbreak of Septoria musiva cankers on Populus balsamifera in a northern Alberta plantation. Four clones of P. balsamifera, five clones of putatively resistant P. deltoides, and one susceptible hybrid poplar clone, Northwest, were inoculated with seven isolates identified as S. musiva. Four of the isolates were from P. balsamifera in Alberta and the others were from P. deltoides in Quebec. Results indicated that disease severity was similar for Alberta and Quebec isolates (P = 0.243) and that P. balsamifera had the greatest mean disease severity (x-bar = 4.20), P. deltoides had the lowest (x-bar = 2.76), and Northwest was intermediate (x-bar = 3.45). A genetic analysis comparing six polymorphic polymerase chain reaction restriction fragment length polymorphism loci and the mitochondrial small subunit ribosomal DNA sequences of the seven isolates indicated that the Alberta population was made up of at least three distinct genotypes. Canker incidence and age on Northwest and 56 different clones of P. balsamifera in a plantation were recorded. Canker incidence (P = 0.726) and the canker age distributions (P = 0.994) were similar for the two species. In conclusion, contrary to what has been reported in the literature, P. balsamifera appears to be quite susceptible to Septoria canker.
Characterization and Pathogenicity of Fusicladium eriobotryae, the Fungal Pathogen Responsible for Loquat Scab
Authors: Sánchez-Torres P. , Hinarejos R. , Tuset J. J.
Published: October 2009

To characterize the fungal pathogen responsible for loquat scab and establish differences in pathogenicity in loquat, eight strains identified as Fusicladium eriobotryae were isolated from either loquat leaves or fruit showing scab symptoms in Spain. Loquat plants belonging to the cv. Peluche were infected via a newly developed infection system that was based on spraying susceptible loquat plants with fungal spore suspensions, keeping the plants for 1 week in 100% humidity, and then transferring the plants to ambient relative humidity in greenhouses. Scab symptoms were analyzed and pathogenic characterization of all F. eriobotryae strains revealed different degrees of aggressiveness. Based on infection progression and severity of scab symptoms, strain ST1 was confirmed as the most aggressive in cultivars in the Mediterranean region. Strain ST1, which even caused chlorotic spots in loquat stems, is so aggressive it can be used to identify highly resistant cultivars using this in vivo system. Molecular characterization of internal transcribed spacer ribosomal DNA and, particularly, the glyceraldehyde 3-phosphate dehydrogenase gene, clearly distinguished loquat strains from Venturia inaequalis. Moreover, random amplified polymorphic DNA (RAPD) and microsatellite-primed polymerase chain reaction techniques were used to qualitatively discriminate between species and report the variations within fungal populations. Molecular variability was checked by comparing all the different strains and enabled the specific identification of F. eriobotryae. Although no association was observed between any pattern and phenotypic traits, such as aggressiveness, RAPD provided a specific profile that allowed fungal identification.
Stream Monitoring for Detection of Phytophthora ramorum in Oregon Tanoak Forests
Authors: Sutton W. , Hansen E. M. , Reeser P. W. , Kanaskie A.
Published: October 2009

Stream monitoring using leaf baits for early detection of Phytophthora ramorum has been an important part of the Oregon Sudden Oak Death (SOD) program since 2002. Sixty-four streams in and near the Oregon quarantine area in the southwest corner of the state were monitored in 2008. Leaves of rhododendron (Rhododendron macrophyllum) and tanoak (Lithocarpus densiflorus) were placed in mesh bags, and bags were floated in streams. Leaf baits were exchanged every 2 weeks throughout the year. Leaves were assayed by isolation on selective medium and by multiplex rDNA internal transcribed spacer polymerase chain reaction (ITS PCR). The two methods gave comparable results, but multiplex PCR was more sensitive. P. ramorum was regularly recovered at all seasons of the year from streams draining infested sites 5 years after eradication treatment. In streams with lower inoculum densities, recovery was much higher in summer than in winter. P. ramorum was isolated from streams in 23 watersheds. When P. ramorum was detected, intensive ground surveys located infected tanoaks or other host plants an average of 306 m upstream from the bait station. P. ramorum was isolated from stream baits up to 1,091 m from the probable inoculum source.
Neofusicoccum spp. Associated with Stem Canker and Dieback of Blueberry in Chile
Authors: Espinoza J. G. , Briceño E. X. , Chávez E. R. , Úrbez-Torres J. R. , Latorre B. A.
Published: October 2009

Blueberry (Vaccinium spp.) plantings have significantly increased in Chile during the last decade and, currently, over 10,700 ha are cultivated throughout the country. Among other diseases, stem canker and dieback has been frequently observed in commercial plantations with incidences between 15 and 45%. The aim of this study was to identify and characterize Neofusicoccum spp. causing stem canker and dieback of blueberry in Chile. Three species, N. arbuti, N. australe, and N. parvum, were identified based on colony and conidia morphology, and nucleotide sequence analysis of the internal transcribed spacer (ITS) region (ITS1-5.8S-ITS2). These Neofusicoccum spp. were found alone or coexisting with Pestalotiopsis spp., Truncatella spp., or Phomopsis spp. Koch's postulates showed all Neofusicoccum spp. isolated from infected plants to be pathogenic when inoculated on blueberry fruit and twigs using both mycelia and conidia suspension. All blueberry cultivars tested, including, Brigitta, Bluecrop, Brightwell, Duke, Elliott, Misty, and O'Neal, were susceptible to Neofusicoccum spp. infection. Pathogenicity tests showed N. parvum to be the most virulent species and Elliott to be the most susceptible cultivar. This report represents the first description of N. arbuti, N. australe, and N. parvum as canker-causing agents on blueberry in Chile.
First Report of Soybean yellow mottle mosaic virus in Soybean in North America
Authors: Li S. , Moon J. S. , Lee S. H. , Domier L. L.
Published: October 2009

Soybean yellow mottle mosaic virus (SYMMV) is a soybean-infecting virus recently discovered in Korea that initially induces bright yellow mosaic on leaves followed by stunting and reduced growth of older leaves (1). Nucleotide sequence analysis of genomic RNA of the Korean SYMMV isolate suggested that the virus is a new member of the genus Carmovirus in the family Tombusviridae. To determine whether SYMMV is present in the United States, single leaflets were collected without regard for symptoms from 7 to 10 plants in each of 136 plots in August 2008 from a research field in Stoneville, MS that contained 16 plant introductions (including five from Korea) and ‘Williams 82’. Samples were grouped into 10 pools of 100 leaves from which total RNA was extracted with the Qiagen RNeasy Plant Mini Kit (Germantown, MD), reverse transcribed, and amplified with SuperScript III Platinum SYBR Green One-Step Quantitative Real-time Reverse Transcriptase-PCR Kit (Invitrogen, Carlsbad, CA) and two pairs of oligonucleotide primers (5′-CGTCTGCCAGGGTTTAATACTA-3′, and 5′-GATTAGCATGTCAGGGTGGTCG-3′; and 5′-ACTGAGTCCCCTGCTTAT-3′ and 5′-CATCACTAGCGTCYGGATCA-3′) that were designed from regions conserved between SYMMV and Cowpea mottle virus (CPMoV; a related and seed-transmitted carmovirus). Six 100-leaflet pools were positive with both primer sets and four pools were negative with both primer sets. Total RNA extracted from one positive pool was reverse transcribed using SuperScript II reverse transcriptase and a primer complementary to nt 4,000 to 4,009 of the SYMMV genome and amplified using iProof DNA polymerase (Bio-Rad, Hercules, CA) as two overlapping DNA fragments using primers corresponding to nt 1 to 21 and complementary to nt 3,483 to 3,508 and corresponding to nt 3,366 to 3,391 and complementary to nt 4,000 to 4,009. DNA fragments were sequenced using a BigDye Terminator Cycle Sequencing Kit and ABI 3730XL capillary sequencers (Applied Biosystems, Foster City, CA). The 4,009-nt sequence of the Mississippi SYMMV isolate (GenBank Accession No. FJ707484) was 96% identical to the Korean SYMMV isolate and 65% identical to CPMoV. Because of the sampling techniques used, it was not possible to associate SYMMV-positive plants with disease symptoms in Mississippi. To our knowledge, this is the first report of SYMMV in North America.

Reference: (1) M. Nam et al. Online publication. doi:10.1077/s00705-009-0480. Arch. Virol., 2009.
Appearance of Powdery Mildew of Wheat Caused by Blumeria graminis f. sp. tritici on Pm17-Bearing Cultivars in North Carolina
Authors: Cowger C. , Parks R. , Marshall D.
Published: October 2009

Pm17 is a gene for resistance to powdery mildew caused by Blumeria graminis (DC.) E.O. Speer f. sp. tritici. The gene was first confirmed in the wheat-rye translocation cultivar Amigo (1). In Amigo, the translocation is T1AL-1RS and the 1RS arm has the gene Pm17. In the mid-Atlantic United States, at least two widely deployed soft red winter wheat (Triticum aestivum L.) cultivars, McCormick (2) and Tribute (3), possess Pm17 inherited from Amigo. Before 2009, low frequencies of mostly intermediate virulence to Pm17 were detected among isolates from research plots of highly susceptible cultivars (4), but Pm17-bearing cultivars remained immune to mildew in the field. In April 2009, moderately severe powdery mildew was observed for the first time throughout plots of McCormick, Tribute, and other cultivars in both Kinston and Raleigh, NC. At Kinston, Pm17 virulence was observed at two research sites, separated by approximately 10 km, throughout plots of Amigo, McCormick, Tribute, and the hard red winter wheat cultivar TAM 303, which also contains Pm17. In the same month, virulence to Pm17 was observed in Raleigh throughout rows and plots of Amigo and TAM 303. In Kinston and Raleigh, ratings of powdery mildew severity on the Pm17-containing cultivars were 4 or 5 on a scale of 0 to 9, with 0 being the absence of mildew pustules and 9 the most severe mildew infection. Mildew was observed on leaves of all ages. Mildewed leaves were collected from field plots of all four Pm17-bearing cultivars, and an assay to confirm Pm17 virulence was conducted in the laboratory. Mixed-isolate cultures were derived from the leaves and a detached-leaf assay was performed using Amigo, which is the standard Pm17 differential (4). All tested cultures were fully to moderately virulent on Pm17 and all were fully virulent on the susceptible control Chancellor. In the field, chasmothecia (sexual fruiting bodies) were observed on Pm17-bearing cultivars. Together with the quantitatively varying Pm17 virulence detected in the laboratory assay, this suggests that multiple strains of Pm17-virulent B. graminis f. sp. tritici may be present in the field, although that has not yet been demonstrated. Pm17 has protected wheat from powdery mildew over a substantial area in the mid-Atlantic United States. The loss of Pm17 is the most important virulence shift in the U.S. wheat powdery mildew population since Pm4a became ineffective around 2002. Isolates virulent to Pm17 can be expected to appear and multiply in wheat-producing states of the mid-Atlantic United States, including Delaware, Maryland, Virginia, North Carolina, South Carolina, and Georgia. Thus, the urgency of developing and releasing wheat cultivars with other sources of effective mildew resistance is heightened.

References: (1) B. Friebe et al. Euphytica 91:59, 1996. (2) C. A. Griffey et al. Crop Sci. 45:416, 2005. (3) C. A. Griffey et al. Crop Sci. 45:419, 2005. (4) R. Parks et al. Plant Dis. 92:1074, 2008.
First Report of Bacterial Leaf Scorch Caused by Xylella fastidiosa on Southern Highbush Blueberry in Florida
Authors: Harmon P. F. , Hopkins D. L.
Published: October 2009

In May of 2008, samples of southern highbush blueberry (interspecific Vaccinium corymbosum hybrids) exhibiting marginal leaf necrosis were collected at a farm in Interlachen, FL. The cv. Star showed severe leaf scorch, partial defoliation, and a generally unthrifty growth structure of many thin twigs as has been observed in Georgia (1). The block of Star plants was approximately 10 years old and incidence of the disease was 100%. The grower reported the planting had become increasingly unproductive in the most recent 4 to 5 years. Plants of the cv. Windsor also showed scorch symptoms and yellow-to-red discoloration of leaves. Proportionally fewer Windsor plants showed disease symptoms than Star plants and the disease was not as severe on this cultivar on the basis of visual estimates at the time. Each sample consisted of 5 to 10 cuttings of spring wood with attached leaves showing marginal necrosis taken from a single plant. Three samples, two from Star plants and one from a Windsor plant, were divided into two subsamples each. One subsample was submitted to Agdia for Xylella fastidiosa double-antibody sandwich (DAS)-ELISA assay (TSE XF; Agdia Inc., Elkhart IN). All three samples were reported as positive for X. fastidiosa by DAS-ELISA. A number of asymptomatic plants from this farm and other additional farms were tested in the same manner and results were negative. The other subsample was used for isolation of the causal bacterium. Petioles and main veins from symptomatic leaves were surface disinfested in 1% sodium hypochlorite, cut into segments (0.5 cm), and squeezed with forceps or pliers. Sap that exuded from the segment was blotted directly onto periwinkle wilt medium (2). Bacterial colonies consistent in morphology with X. fastidiosa that were DAS-ELISA positive were obtained from all three samples. One isolate from each sample was inoculated into four Star plants each with the pin-pricking method (3). Leaf scorch symptoms were first observed 8 weeks after inoculation. By 12 weeks after inoculation, all plants inoculated with the three isolates had developed symptoms, including defoliation. Plants inoculated without bacteria showed no symptoms. X. fastidiosa was reisolated from symptomatic plants. Bacterial leaf scorch is an important emerging disease that threatens the southern highbush blueberry industry in the south. On certain cultivars like Star, the potential to reduce yield appears to be great. Differences between cultivars are likely, but have not yet been explored. Additional research is needed into the epidemiology of the disease and potential vectors of pathogen transmission.

References: (1) C. Chang et al. HortScience 44:413, 2009. (2) M. Davis et al. Curr. Microbiol. 6:309, 1981. (3) D. L. Hopkins et al. Phytopathology 75:713, 1985.
First Report of Plasmodiophora brassicae on Rapeseed in the Grand Duchy of Luxembourg
Authors: Desoignies N. , Eickermann M. , Delfosse P. , Kremer F. , Godart N. , Hoffmann L. , Legrève A.
Published: October 2009

Rapeseed (Brassica napus L.) is the third most important crop after wheat and barley in the Grand Duchy of Luxembourg. Since 2005, clubroot symptoms in this crop have been reported by farmers in the Gutland Region. In February 2009, plants of the hybrid rapeseed cv. Exocet, with stunted growth, yellow leaves, and club-shaped roots, were sampled from a field in Oberkorn village near Differdange. Microscopic observations of the rapeseed root fragments revealed the presence of the three life stages characteristic of Plasmodiophora brassicae Woronin. Plasmodia and zoosporangia were observed in the root hairs and resting spores were present in root galls. Individual spores were 2 to 3 μm in diameter. Total DNA was extracted from the root galls with a FAST DNA Kit (MP Biomedicals, Irvine, CA). The internal transcribed spacer region (ITS) and 5.8S gene of the rDNA region were amplified with ITS5 and ITS4 primers as described by White et al. (2) and part of this region was sequenced. A BLASTn search in GenBank revealed that the sequence closely resembled (98% identity) sequences of P. brassicae (Genbank Accession No. EF195335) from an isolate of the pathogen from Switzerland. To confirm the presence of the pathogen, seeds of the susceptible ecotype cvi-0 of Arabidopsis thaliana were grown in a soil sample (1 liter) collected near the infected rapeseed plants. After 55 days of growth in a glasshouse at 15 to 20°C, the roots of 11 plants were analyzed. Two showed clear clubroot symptoms and four others exhibited small swellings. The remaining five plants were symptomless, but plasmodia and zoosporangia were found in root hair cells. Clubroot caused by P. brassicae has previously been described on B. napus and other crucifers (1). To our knowledge, this is the first report of clubroot disease caused by P. brassicae in Luxembourg. Because its presence has since been observed in new fields in the Gutland Region and because of the ability of the pathogen to survive for a long period in the soil, this disease could represent a severe threat for cropping of Brassicaceae in Luxembourg and neighboring countries.

References: (1) I. R. Crute et al. Plant Breed. Abstr. 50:91, 1980. (2) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al., eds. Academic press: San Diego, 1990.
First Report of Leveillula taurica Causing Powdery Mildew on Pepper in Maryland
Authors: Jones R. W. , Stommel J. R. , Wanner L. A.
Published: October 2009

Pepper plants in large experimental plots in Beltsville, MD developed widespread powdery mildew during the late summer of 2008. Infection was observed in a diversity of accessions that included Capsicum annuum, C. baccatum, C. chinense, and C. frutescens (2). The C. annuum accessions included culinary bell pepper cultivars and breeding lines as well as a diverse collection of ornamental breeding lines, heirlooms, and land races. Significant leaf damage occurred and led to partial defoliation. Extensive coverage of the abaxial surface by white patches of conidia was noted, along with chlorotic regions on the adaxial surface. Conidia were borne singly and were apically tapered, measuring 65.2 ± 3.2 × 14.9 ± 1.9 μm. Cleistothecia were not found on infected leaves (3). PCR amplification of the internal transcribed spacer (ITS) region using ITS1-2 primers yielded a band that was cloned and sequenced (4). The pathogen was identified as Leveillula taurica based on 100% homology to GenBank Accession No. AY912077. Multiple chili pepper and bell pepper plants were inoculated with conidia from an infected bell pepper plant by placement in an enclosed spore deposition chamber for 1 week, with the infected plant suspended over the test plants. Signs of powdery mildew appeared only on inoculated plants. DNA samples from these inoculated plants were analyzed and verified as L. taurica (a sequence was deposited as GenBank No. GQ167201). A second set of inoculations using the newly infected plants confirmed results of the first test, with mildew developing only on inoculated pepper plants. This disease is new to the mid-Atlantic Region of the United States. It has been reported in greenhouse peppers growing in Ontario, Canada where it has become a recurring problem requiring fungicide intervention (1). Given the wide host range of L. taurica and the systemic nature of infections, it is likely that the fungus has become established in Maryland on perennial host plants.

References: (1) R. Cerkauskas. Plant Dis. 83:781, 1999. (2) V. de Souza. Plant Pathol. 52:613, 2003. (3) C. Little. Plant Dis. 90:1358, 2006. (4) G. Saenz. Can. J. Bot. 77:150, 1999.
Role of biofilm formation in the persistent colonization of Haemophilus influenzae in children from northern India
Authors: Sekhar Sasanka , Kumar Rajesh , Chakraborti Anuradha
Published: October 2009

The human nasopharynx is a major ecological niche for Haemophilus influenzae colonization. Establishment of infection is critically dependent on the persistence of bacteria in the nasopharynx. Various factors are presumed to mediate this persistence and the influence of biofilm formation has been under scrutiny for a long time. In a prospective population-based study, the nasopharyngeal colonization pattern of 250 children <2 years old was traced to gain further insights into the phenomenon. The association between biofilm formation and persistence was delineated by quantitative biofilm assay, while the true nature of biofilm formers was further evaluated by electron microscopy studies. H. influenzae isolates obtained in this study, when analysed by phenotypic and genotypic means, revealed a clonal distribution of strains within the population. The biofilm formation ability of the isolates was found to be significantly associated with bacterial persistence (P<0.001). The isolates having biofilm formation ability were found to be 7.1 times more likely to persist in the nasopharynx than non-biofilm formers. This study provides direct evidence indicating the intricate relationship between biofilm formation and the persistence of bacteria. Our results emphasize the need to evaluate the potential for biofilm formation before designing preventive and therapeutic strategies.
Antifungal activity of the clove essential oil from Syzygium aromaticum on Candida, Aspergillus and dermatophyte species
Authors: Pinto Eugénia , Vale-Silva Luís , Cavaleiro Carlos , Salgueiro Lígia
Published: October 2009

The composition and antifungal activity of clove essential oil (EO), obtained from Syzygium aromaticum, were studied. Clove oil was obtained commercially and analysed by GC and GC-MS. The EO analysed showed a high content of eugenol (85.3 %). MICs, determined according to Clinical and Laboratory Standards Institute protocols, and minimum fungicidal concentration were used to evaluate the antifungal activity of the clove oil and its main component, eugenol, against Candida, Aspergillus and dermatophyte clinical and American Type Culture Collection strains. The EO and eugenol showed inhibitory activity against all the tested strains. To clarify its mechanism of action on yeasts and filamentous fungi, flow cytometric and inhibition of ergosterol synthesis studies were performed. Propidium iodide rapidly penetrated the majority of the yeast cells when the cells were treated with concentrations just over the MICs, meaning that the fungicidal effect resulted from an extensive lesion of the cell membrane. Clove oil and eugenol also caused a considerable reduction in the quantity of ergosterol, a specific fungal cell membrane component. Germ tube formation by Candida albicans was completely or almost completely inhibited by oil and eugenol concentrations below the MIC values. The present study indicates that clove oil and eugenol have considerable antifungal activity against clinically relevant fungi, including fluconazole-resistant strains, deserving further investigation for clinical application in the treatment of fungal infections.
Septic arthritis due to Roseomonas gilardii in an immunocompetent adolescent
Authors: Fanella Sergio , Schantz Daryl , Karlowsky James , Rubinstein Ethan
Published: October 2009

The genus Roseomonas comprises groups of slow-growing, Gram-negative coccobacilli, which only infrequently cause infection in humans. When identified, they are associated with immunocompromised adults, often causing bacteraemia. Due to their rarity, members of this genus can be overlooked or misidentified using automated laboratory identification systems. We report on an immunocompetent adolescent patient who developed septic arthritis due to Roseomonas gilardii following surgery for a sports injury. The isolate was initially misidentified as Bordetella bronchiseptica using the Vitek 2 system, but confirmed as R. gilardii based on 16S rRNA gene sequencing. To the best of our knowledge, this is the first case of a healthy paediatric patient with septic arthritis due to R. gilardii.
Characterization of multidrug-resistant and extended-spectrum β-lactamase-producing Klebsiella pneumoniae strains from Malaysian hospitals
Authors: Lim King Ting , Yeo Chew Chieng , Md Yasin Rohani , Balan Ganeswrie , Thong Kwai Lin
Published: October 2009

The emergence of multidrug-resistant (MDR) and extended-spectrum β-lactamase (ESBL)-producing Klebsiella pneumoniae poses a serious antibiotic management problem as resistance genes are easily transferred from one organism to another. Fifty-one strains of K. pneumoniae isolated from sporadic cases in various hospitals throughout Malaysia were analysed by antimicrobial susceptibility testing, PCR detection of ESBL-encoding genes and DNA fingerprinting. Although 27 of the 51 K. pneumoniae strains were MDR (i.e. resistant to three or more classes of antibiotics), the majority of the strains (98 %) were sensitive to imipenem. PCR detection using ESBL gene-specific primers showed that 46 of the K. pneumoniae strains harboured bla
SHV, 19 harboured bla
CTX-M, 5 harboured bla
OXA-1 and 4 harboured bla
TEM-1. Class 1 integron-encoded intI1 integrase was detected in 21 of the 51 K. pneumoniae strains and amplification of the integron 5′CS region showed the presence of several known antibiotic resistance gene cassettes of various sizes. Results of conjugation and transformation experiments indicated that some of the ESBL-encoding genes (i.e. bla
SHV, bla
CTX-M and bla
TEM-1) were transmissible and were likely plasmid-encoded. DNA fingerprinting using PFGE and PCR-based methods indicated that the 51 K. pneumoniae strains were genetically diverse and heterogeneous.
Burkholderia cepacia keratitis with endophthalmitis
Authors: Örnek Kemal , Özdemir Mehmet , Ergin Ahmet
Published: October 2009

Keratitis with endophthalmitis of the right eye occurred in a 78-year-female following a complicated cataract surgery. Prompt intravitreal vancomycin and ceftazidime with topical fortified tobramycin and cefazolin treatments were started. The corneal, aqueous and vitreous cultures grew a Burkholderia cepacia complex (Bcc) strain on the fourth day. Restriction fragment length polymorphism analysis of the recA amplicon revealed B. cepacia genomovar I. The organism was found to be susceptible to ceftazidime, ciprofloxacin and ofloxacin. Topical ciprofloxacin was given immediately. At day 10, the pain relieved and the clinical condition of the patient improved with resolution of the purulent discharge, severe circumcorneal congestion and chemosis. The size of the corneal abscess and anterior chamber exudation decreased. The Bcc should be included among the bacterial species that may cause keratitis following intraocular surgeries.
Corynebacterium diphtheriae as an emerging pathogen in nephrostomy catheter-related infection: evaluation of traits associated with bacterial virulence
Authors: Gomes Débora L. R. , Martins Carlos A. S. , Faria Lúcia M. D. , Santos Louisy S. , Santos Cintia S. , Sabbadini Priscila S. , Souza Mônica C. , Alves Gabriela B. , Rosa Ana C. P. , Nagao Prescilla E. , Pereira Gabriela A. , Hirata Raphael , Mattos-Guaraldi Ana L.
Published: October 2009

Corynebacterium diphtheriae still represents a global medical challenge, particularly due to the significant number of individuals susceptible to diphtheria and the emergence of non-toxigenic strains as the causative agents of invasive infections. In this study, we characterized the clinical and microbiological features of what we believe to be the first case of C. diphtheriae infection of a percutaneous nephrostomy catheter insertion site in an elderly patient with a fatal bladder cancer. Moreover, we demonstrated the potential role of adherence, biofilm formation and fibrin deposition traits in C. diphtheriae from the catheter-related infection. Non-toxigenic C. diphtheriae isolated from the purulent discharge (named strain BR-CAT5003748) was identified by the API Coryne system (code 1 010 324) and a multiplex PCR for detection of dtxR and tox genes. Strain BR-CAT5003748 showed resistance to oxacillin, ceftazidime and ciprofloxacin. In experiments performed in vitro, the catheter isolate was classified as moderately hydrophobic and as moderately adherent to polystyrene surfaces. Glass provided a more effective surface for biofilm formation than polystyrene. Micro-organisms adhered to (>1.5×106 c.f.u.) and multiplied on surfaces of polyurethane catheters. Microcolony formation (a hallmark of biofilm formation) and amorphous accretions were observed by scanning electron microscopy on both external and luminal catheter surfaces. Micro-organisms yielded simultaneous expression of localized adherence-like and aggregative-like (LAL/AAL) adherence patterns to HEp-2 cells. Interestingly, the coagulase tube test resulted in the formation of a thin layer of fibrin embedded in rabbit plasma by the non-toxigenic BR-CAT5003748 strain. In conclusion, C. diphtheriae should be recognized as a potential cause of catheter-related infections in at-risk populations such as elderly and cancer patients. LAL/AAL strains may be associated with virulence traits that enable C. diphtheriae to effectively produce biofilms on catheter surfaces. Biofilm formation and fibrin deposition could have contributed to the persistence of C. diphtheriae at the infected insertion site and the obstruction of the nephrostomy catheter.
Rapid detection of anti-hepatitis A virus neutralizing antibodies in a microplate enzyme immunoassay
Authors: Azizi Ali , Sirskyj Danylo , Weltzin Richard , Anderson David E. , Diaz-Mitoma Francisco
Published: October 2009

The slow growth of hepatitis A virus (HAV) in cell culture is one of the primary pitfalls in the development of sensitive and rapid methods for the detection and quantification of HAV and associated neutralizing antibodies. Currently, in vitro assays frequently require 8 days or more to detect and quantify the presence of HAV neutralizing antibodies. This study describes a rapid immunoassay that allowed the detection of anti-HAV neutralizing antibodies in only 3 days. This microplate-based enzymic assay may be applicable in virological diagnostics, in evaluating the immunogenicity of HAV vaccines and in quantifying neutralizing antibodies during the course of HAV infection.
Molecular characterization of extended-spectrum β-lactamase-producing Escherichia coli isolates from chickens in Henan Province, China
Authors: Yuan Li , Liu Jian-Hua , Hu Gong-Zheng , Pan Yu-Shan , Liu Zhi-Ming , Mo Juan , Wei Yong-Jun
Published: October 2009

Extended-spectrum β-lactamase (ESBL)-producing Escherichia coli has spread rapidly worldwide and poses a serious threat to human and animal health. This study collected 51 non-replicate E. coli isolates from 14 different chicken farms in Henan Province in China from December 2007 to August 2008. The prevalence of ESBL-producing E. coli, molecular characterization of the ESBL-related bla genes, including bla
TEM, bla
SHV and bla
CTX-M, and the susceptibilities of these bacteria to various antimicrobial agents were determined. Thirty-one of the 51 isolates were positive for an ESBL phenotype and 29 of these isolates carried one or more bla genes. Twenty-two isolates harboured bla
TEM genes and 15 isolates carried bla
CTX-M genes (one CTX-M-14, three CTX-M-24 and 11 CTX-M-65). One isolate carried bla
TEM
-57; the remaining bla
TEM isolates carried bla
TEM-1 with one silent nucleotide base variation (T18C). We believe that this is the first study to report TEM-57 in E. coli isolates. All isolates harbouring bla
CTX-M-24 and bla
CTX-M-14 and five of the bla
CTX-M-65 isolates also harboured the bla
TEM-1 gene. To our knowledge, this study is the first to describe detection of CTX-M-65-producing E. coli isolated from chickens. None of the isolates contained the bla
SHV gene. Conjugation experiments demonstrated that bla
CTX-M and bla
TEM genes could be transferred to E. coli DH5α. The results indicate that ESBL frequency has reached an alarming level in chicken isolates in China, with TEM-1 and CTX-M-65 enzymes being the two predominant β-lactamases detected.
A safety and feasibility study comparing an intermittent high dose with a daily standard dose of liposomal amphotericin B for persistent neutropenic fever
Authors: Ellis Michael , Bernsen Roos , Ali-Zadeh Hussein , Kristensen Jörgen , Hedström Ulla , Poughias Lazaros , Bresnik Mark , Al-Essa Awad , A. Stevens David
Published: October 2009

A high intermittent dose regimen (group A: 10 mg kg−1 on day 1, 5 mg kg−1 on days 3 and 6) was compared with standard dosing (group B: 3 mg kg−1 per day for 14 days) of liposomal amphotericin B (LAB) for empirical treatment of persistent febrile neutropenia. A total cumulative dose of 1275 mg (group A) and 2800 mg (group B) was administered. Infusion-related adverse drug events, mainly rigors/chills, occurred more frequently with group A (11/45, 24 % infusions) than with group B (12/201, 6 % infusions) (P=0.002), which extended the mean infusion time by 20 min (P=0.001). Creatinine levels were similar in the two regimens: the A : B ratio of the area under the curve for creatinine (AUCCREATININE) for days 2–7 was 1.09 (P=0.27) and for days 2–14 was 1.05 (P=0.51). Rises in creatinine were mild (clinical toxicity criteria 1) in all patients with elevations. Hypokalaemia tended to be less severe in group A with a lower proportion of hypokalaemic days [57/143 (39 %) vs 80/137 (58 %), P=0.21], a higher AUCPOTASSIUM (A : B ratio of 1.06, P=0.12), a lower proportion of patients with hypokalaemia at the end of study (10 vs 61 %, P=0.01) and fewer potassium-supplemented days [12/210 (6 %) vs 41/210 (19.5 %), P<0.1]. There were mildly elevated median levels of serum bilirubin, alanine aminotransferase, aspartate aminotransferase and alkaline phosphatase, which were similar for the two regimens and were usually associated with other co-existing co-morbid conditions. The AUC for these enzymes was also similar in the two groups. No patient had discontinuation of the study drug due to toxicity. Composite success was identical for each regimen (11/15 patients, 73 %). Three of the fifteen patients in group B and none in group A developed invasive fungal infections (IFIs). β-d-Glucan levels were similar in both groups for patients without an IFI [AUCGLUCAN of 362 and 683 (P=0.36) for groups A and B, respectively]. The rate of defervescence was similar for each regimen (P=0.75). This feasibility study suggests that a short intermittent high-dose course of 10/5/5 mg LAB kg−1 on days 1, 3 and 6 may be as safe and effective as a standard 14 day course of 3 mg kg−1 per day, with drug-acquisition cost savings and reduced drug exposure. A larger study is indicated for confirmation of this.
Studying bacterial infections through culture-independent approaches
Authors: Rogers Geraint B. , Carroll Mary P. , Bruce Kenneth D.
Published: October 2009

The ability to characterize accurately the cause of infection is fundamental to effective treatment. The impact of any antimicrobial agents used to treat infection will, however, always be constrained by both the appropriateness of their use and our ability to determine their effectiveness. Traditional culture-based diagnostic microbiology is, in many cases, unable to provide this information. Molecular microbiological approaches that assess the content of clinical samples in a culture-independent manner promise to change dramatically the types of data that are obtained routinely from clinical samples. We argue that, in addition to the technical advance that these methodologies offer, a conceptual advance in the way that we reflect on the information generated is also required. Through the development of both of these advances, our understanding of infection, as well as the ways in which infections can be treated, may be improved. In the analysis of the microbiological content of certain clinical samples, such as blood, cerebrospinal fluid, brain and bone biopsy, culture-independent approaches have been well documented. Herein, we discuss how extensions to such studies can shape our understanding of infection at the many sites of the human body where a mixed flora, or in more ecological terms, a community of microbes, is present. To do this, we consider the underlying principles that underpin diagnostic systems, describe the ways in which these systems can be applied to community characterization, and discuss the significance of the data generated. We propose that at all locations within the human body where infection is routinely initiated within the context of a community of microbes, the same principles will apply. To consider this further, we take insights from areas such as the gut, oral cavity and skin. The main focus here is understanding respiratory tract infection, and specifically the infections of the cystic fibrosis lung. The impact that the use of culture-independent, molecular analyses will have on the way we approach the treatment of infections is also considered.
Hepatitis delta virus proteins repress hepatitis B virus enhancers and activate the alpha/beta interferon-inducible MxA gene
Authors: Williams Virginie , Brichler Ségolène , Radjef Nadjia , Lebon Pierre , Goffard Anne , Hober Didier , Fagard Remi , Kremsdorf Dina , Dény Paul , Gordien Emmanuel
Published: October 2009

Co-infection and superinfection of hepatitis B virus (HBV) with hepatitis delta virus (HDV) leads to suppression of HBV replication both in patients and in animal and cellular models. The mechanisms behind this inhibition have not previously been explored fully. HBV replication is governed by four promoters and two enhancers, Enh1 and Enh2. Repression of these enhancers has been reported to be one of the main mechanisms of HBV inhibition. Moreover, in a previous study, it has been demonstrated that alpha interferon (IFN-α)-inducible MxA protein inhibits HBV replication. HDV encodes two proteins, p24 and p27. p27 was shown to activate several heterologous promoters, including HBV promoters. In an attempt to analyse the mechanisms of HBV inhibition by HDV, the question was raised whether HDV proteins could act directly by repressing HBV enhancers, and/or indirectly by activating the MxA gene. This issue was addressed in a co-transfection model in Huh-7 cells, using p24- or p27-expressing plasmids along with Enh1, Enh2, HBV and MxA promoter–luciferase constructs. Enh1 and Enh2 were strongly repressed, by 60 and 80 % and 40 and 60 %, by p24 and p27, respectively. In addition, p27 was responsible for threefold activation of the MxA promoter and potentiation of IFN-α on this promoter. MxA mRNA quantification and a virus yield reduction assay confirmed these results. In conclusion, this study shows that HDV proteins inhibit HBV replication by trans-repressing its enhancers and by trans-activating the IFN-α-inducible MxA gene.
Toll-like receptor 7-induced immune response to cutaneous West Nile virus infection
Authors: Welte Thomas , Reagan Krystle , Fang Hao , Machain-Williams Carlos , Zheng Xin , Mendell Nicole , Chang Gwong-Jen J. , Wu Ping , Blair Carol D. , Wang Tian
Published: October 2009

The Toll-like receptor (TLR) 7 response represents a vital host-defence mechanism in a murine model of systemic West Nile virus (WNV) infection. Here, we investigated the role of the TLR7-induced immune response following cutaneous WNV infection. We found that there was no difference in susceptibility to WNV encephalitis between wild-type and TLR7−/− mice upon intradermal injection or infected mosquito feeding. Viral load analysis revealed similar levels of WNV RNA in the peripheral tissues and brains of these two groups of mice following intradermal infection. There was a higher level of cytokines in the blood of wild-type mice at early stages of infection; however, this difference was diminished in the blood and brains at later stages. Langerhans cells (LCs) are permissive to WNV infection and migrate from the skin to draining lymph nodes upon intradermal challenge. Our data showed that WNV infection of TLR7−/− keratinocytes was significantly higher than that of wild-type keratinocytes. Infection of wild-type keratinocytes induced higher levels of alpha interferon and interleukin-1β (IL-1β), IL-6 and IL-12, which might promote LC migration from the skin. Co-culture of naïve LCs of wild-type mice with WNV-infected wild-type keratinocytes resulted in the production of more IL-6 and IL-12 than with TLR7−/− keratinocytes or by cultured LCs alone. Moreover, LCs in the epidermis were reduced in wild-type mice, but not in TLR7−/− mice, following intradermal WNV infection. Overall, our results suggest that the TLR7 response following cutaneous infection promotes LC migration from the skin, which might compromise its protective effect in systemic infection.
Bovine leukemia virus can be classified into seven genotypes: evidence for the existence of two novel clades
Authors: Rodriguez Sabrina M. , Golemba Marcelo D. , Campos Rodolfo H. , Trono Karina , Jones Leandro R.
Published: October 2009

Previous studies have classified the env sequences of bovine leukemia virus (BLV) provirus from different locations worldwide into between two and four genetic groupings. These different studies gave unique names to the identified groups and no study has yet integrated all the available sequences. Thus, we hypothesized that many of the different groups previously identified actually correspond to a limited group of genotypes that are unevenly distributed worldwide. To examine this hypothesis, we sequenced the env gene from 28 BLV field strains and compared these sequences to 46 env sequences that represent all the genetic groupings already identified. By using phylogenetic analyses, we recovered six clades, or genotypes, that we have called genotypes 1, 2, 3, 4, 5 and 6. Genotypes 1–5 have counterparts among the sequence groupings identified previously. One env sequence did not cluster with any of the others and was highly divergent when compared with the six genotypes identified here. Thus, an extra genotype, which we named 7, may exist. Similarity comparisons were highly congruent with phylogenetic analyses. Furthermore, our analyses confirmed the existence of geographical clusters.
Myristoylation of the arterivirus E protein: the fatty acid modification is not essential for membrane association but contributes significantly to virus infectivity
Authors: Thaa Bastian , Kabatek Aleksander , Zevenhoven-Dobbe Jessika C. , Snijder Eric J. , Herrmann Andreas , Veit Michael
Published: October 2009

The envelope of equine arteritis virus (EAV) contains two glycoprotein complexes (GP2b/GP3/GP4 and GP5/M) and the small, non-glycosylated E protein. As E is essential for the production of infectious progeny but dispensable for assembly and release of virus-like particles, it probably mediates virus entry into cells, putatively in concert with the GP2b/GP3/GP4 complex. The E protein contains a central hydrophobic domain and a conserved potential site for N-terminal myristoylation, a hydrophobic modification usually pivotal for membrane targeting of the modified protein. Here, it was shown by radiolabelling that E is myristoylated at glycine-2, both in transfected cells as a fusion protein with yellow fluorescent protein (YFP) and in virus particles. Biochemical fractionation revealed that E–YFP with an inactivated acylation site was still completely membrane-bound, indicating that the putative transmembrane domain of E mediates membrane targeting. Confocal microscopy showed that both myristoylated and non-myristoylated E–YFP were localized to the endoplasmic reticulum and Golgi complex, the membranes from which EAV buds. The presence of a myristoylation inhibitor during replication of EAV, whilst completely blocking E acylation, reduced virus titres by 1.5 log10. Similarly, a mutant EAV with non-myristoylatable E grew to a titre five- to sevenfold lower than that of the wild-type virus and exhibited a reduced plaque size. Western blotting of cell-culture supernatants showed that N and M, the major structural proteins of EAV, are released in similar amounts by cells transfected with wild-type and mutant genomes. Thus, E myristoylation is not required for budding of particles and probably has a function during virus entry.
Apoptosis in lymphoid tissues of calves inoculated with non-cytopathic bovine viral diarrhea virus genotype 1: activation of effector caspase-3 and role of macrophages
Authors: Pedrera Miriam , Gómez-Villamandos José C. , Romero-Trevejo José L. , Risalde María A. , Molina Verónica , Sánchez-Cordón Pedro J.
Published: October 2009

The mechanisms responsible for lymphocyte apoptosis in bovine viral diarrhoea have not yet been clarified. Previous work suggests that bovine viral diarrhea virus (BVDV) is only directly responsible for the destruction of a small number of lymphocytes. The aim of this study was to clarify, in vivo, the role of macrophages in lymphocyte destruction through indirect mechanisms linked to the biosynthetic activation of these immunocompetent cells on ileal Peyer's patches, as well as the distribution and quantification of apoptosis. Eight colostrum-deprived calves were inoculated intranasally with a non-cytopathic strain of BVDV genotype 1 and killed in batches of two at 3, 6, 9 and 14 days post-inoculation (p.i.). The progressive depletion of Peyer's patches was found to be due to massive lymphocyte apoptosis, with an increase in cleaved caspase-3 and TUNEL-positive cells. Lymphoid depletion was accompanied, from 3 days p.i., by a significant rise in macrophage numbers both in lymphoid follicles and in interfollicular areas. Some macrophages showed signs of viral infection, together with subcellular changes indicative of phagocyte activation and, in some cases, of secretory activity. However, the number of macrophages that showed positive immunostaining for tumour necrosis factor-α and interleukin-1α, cytokines with a proven ability to induce apoptosis, remained low throughout the experiment in lymphoid follicles, where most apoptotic cells were found. These results thus appear to rule out a major involvement of macrophages and macrophage-secreted chemical mediators in the apoptosis of follicular B lymphocytes during BVDV infection.