Host and environmental reservoirs of infection for bovine digital dermatitis treponemes.

Bovine digital dermatitis (BDD) is a global infectious disease causing lameness of cattle and is responsible for substantial animal welfare issues and economic losses. The causative agents are considered to be spirochetal bacteria belonging to the genus Treponema, which have consistently been identified in BDD lesions worldwide. One potential means of controlling infection is the disruption of transmission; however, the infection reservoirs and transmission routes of BDD treponemes have yet to be elucidated. To address these issues, we surveyed for evidence of BDD treponeme presence in the dairy farm environment, in bovine tissues and in bovine gastrointestinal (GI) tract contents. A total of 368 samples were tested using PCR assays specific for each of three currently recognised, isolated phylotypes of BDD treponemes. All environmental samples, together with insects and GI tract content samples were negative for BDD treponeme DNA from the three phylotypes. However, we identified BDD treponemes in two non-pedal bovine regions: the oral cavity (14.3% of cattle tested) and the rectum (14.8% of cattle tested). Whilst only single phylotypes were detected in the oral cavity, two of the rectal tissues yielded DNA from more than one phylotype, with one sample yielding all three BDD treponeme phylotypes. Whilst it might be considered that direct skin to skin contact may be a major transmission route of BDD treponemes, further studies are required to characterise and determine the potential contribution of oral and rectal carriage to BDD transmission.

PCR improves diagnostic yield from lung aspiration in Malawian children with radiologically confirmed pneumonia.

BACKGROUND: Accurate data on childhood pneumonia aetiology are essential especially from regions where mortality is high, in order to inform case-management guidelines and the potential of prevention strategies such as bacterial conjugate vaccines. Yield from blood culture is low, but lung aspirate culture provides a higher diagnostic yield. We aimed to determine if diagnostic yield could be increased further by polymerase chain reaction (PCR) detection of bacteria (Streptococcus pneumoniae and Haemophilus influenzae b) and viruses in lung aspirate fluid. METHODS: A total of 95 children with radiological focal, lobar or segmental consolidation had lung aspirate performed and sent for bacterial culture and for PCR for detection of bacteria, viruses and Pneumocystis jirovecii. In children with a pneumococcal aetiology, pneumococcal bacterial loads were calculated in blood and lung aspirate fluid. RESULTS: Blood culture identified a bacterial pathogen in only 8 patients (8%). With the addition of PCR on lung aspirate samples, causative pathogens (bacterial, viral, pneumocystis) were identified singly or as co-infections in 59 children (62%). The commonest bacterial organism was S.pneumoniae (41%), followed by H. influenzae b (6%), and the commonest virus identified was adenovirus (16%), followed by human bocavirus (HBoV) (4%), either as single or co-infection. CONCLUSIONS: In a select group of African children, lung aspirate PCR significantly improves diagnostic yield. Our study confirms a major role of S.pneumoniae and viruses in the aetiology of childhood pneumonia in Africa.

Characterization of novel bovine gastrointestinal tract Treponema isolates and comparison with bovine digital dermatitis treponemes.

This study aimed to isolate and characterize treponemes present in the bovine gastrointestinal (GI) tract and compare them with bovine digital dermatitis (BDD) treponemes. Seven spirochete isolates were obtained from the bovine GI tract, which, on the basis of 16S rRNA gene comparisons, clustered within the genus Treponema as four novel phylotypes. One phylotype was isolated from several different GI tract regions, including the omasum, colon, rumen, and rectum. These four phylotypes could be divided into two phylotype pairs that clustered closest with each other and then with different, previously reported rumen treponemes. The treponemes displayed great genotypic and phenotypic diversity between phylotypes and differed considerably from named treponeme species and those recently reported by metagenomic studies of the bovine GI tract. Phylogenetic inference, based on comparisons of 16S rRNA sequences from only bovine treponemes, suggested a marked divergence between two important groups. The dendrogram formed two major clusters, with one cluster containing GI tract treponemes and the other containing BDD treponemes. This division among the bovine treponemes is likely the result of adaptation to different niches. To further differentiate the bovine GI and BDD strains, we designed a degenerate PCR for a gene encoding a putative virulence factor, tlyC, which gave a positive reaction only for treponemes from the BDD cluster.

Healthcare-associated viral gastroenteritis among children in a large pediatric hospital, United Kingdom.

Viruses are the major pathogens of community-acquired (CA) acute gastroenteritis (AGE) in children, but their role in healthcare-associated (HA) AGE is poorly understood. Children with AGE hospitalized at Alder Hey Children's Hospital, Liverpool, UK, were enrolled over a 2-year period. AGE was classified as HA if diarrhea developed > or =48 hours after admission. Rotavirus, norovirus, adenovirus 40/41, astrovirus, and sapovirus were detected by PCR. A total of 225 children with HA-AGE and 351 with CA-AGE were enrolled in the study. HA viral gastroenteritis constituted one fifth of the diarrheal diseases among hospitalized children and commonly occurred in critical care areas. We detected > or =1 virus in 120 (53%) of HA-AGE cases; rotavirus (31%), norovirus (16%), and adenovirus 40/41 (15%) were the predominant viruses identified. Molecular evidence indicated rotaviruses and noroviruses were frequently introduced into the hospital from the community. Rotavirus vaccines could substantially reduce the incidence of HA-AGE in children.

Adenovirus infections within a family cohort in Iran.

BACKGROUND: Adenovirus is one of the most frequent viruses associated with acute respiratory infections (ARI). There is limited information of its transmission within the community. METHODS: Cohorts of 50 families with > or =two children were visited weekly for 2 months to ascertain the presence ARI in Rasht, Iran. Nasopharyngeal swabs were obtained from symptomatic participants and at 3-4-day intervals to assess the duration of adenovirus shedding. Adenoviruses were identified by PCR and adenovirus positive amplicons were subjected to DNA sequencing. RESULTS: Thirty-three (35%) of 94 ARI episodes in children and 8 (27%) of 30 episodes in adults were due to adenovirus (not significant, NS). 25/50 (50%) families had adenovirus infections. Children had more infections than adults, were more likely to develop symptoms if there was a symptomatic case within the household and episodes had a longer duration (P < 0.05). Adenoviruses were recovered for a median of 11 (interquartile range 5-26) days of follow up in children and 7 (2-20) days in adults (NS). Adenovirus-7 was the most frequent serotype (12 families), followed by adenovirus-6 (5 families), adenovirus-1 and 2 (4 families each), and adenovirus-5 (3 families). Both adenovirus-5 and 7 amplicons fell into two clusters. No mutations were observed during transmission within a family. CONCLUSION: A substantial proportion of ARI in the community are due to adenovirus with further transmission within the family. Children > or =2 years experienced a higher proportion of infections than younger children and adults. Viral shedding was more prolonged in children and adenovirus-7 and 5 predominated with several clusters co-circulating in the same season.

The genome of Burkholderia cenocepacia J2315, an epidemic pathogen of cystic fibrosis patients.

Bacterial infections of the lungs of cystic fibrosis (CF) patients cause major complications in the treatment of this common genetic disease. Burkholderia cenocepacia infection is particularly problematic since this organism has high levels of antibiotic resistance, making it difficult to eradicate; the resulting chronic infections are associated with severe declines in lung function and increased mortality rates. B. cenocepacia strain J2315 was isolated from a CF patient and is a member of the epidemic ET12 lineage that originated in Canada or the United Kingdom and spread to Europe. The 8.06-Mb genome of this highly transmissible pathogen comprises three circular chromosomes and a plasmid and encodes a broad array of functions typical of this metabolically versatile genus, as well as numerous virulence and drug resistance functions. Although B. cenocepacia strains can be isolated from soil and can be pathogenic to both plants and man, J2315 is representative of a lineage of B. cenocepacia rarely isolated from the environment and which spreads between CF patients. Comparative analysis revealed that ca. 21% of the genome is unique in comparison to other strains of B. cenocepacia, highlighting the genomic plasticity of this species. Pseudogenes in virulence determinants suggest that the pathogenic response of J2315 may have been recently selected to promote persistence in the CF lung. The J2315 genome contains evidence that its unique and highly adapted genetic content has played a significant role in its success as an epidemic CF pathogen.

Morphology, calcium signaling and mechanical activity in human ureter.

PURPOSE: We determined the mechanisms of calcium signaling in the human ureter, and the relationship to peristaltic contractions and bundular structure in living tissue, thereby advancing the understanding of ureteral function in health and obstruction and reflux. MATERIALS AND METHODS: Confocal imaging of 31 ureters was performed and simultaneous force and calcium measurements were made. Immunohistochemistry and Western blotting were also performed. RESULTS: Confocal imaging showed a 3-dimensional network of smooth muscle bundles with no defined longitudinal or circular layers. Fast propagating Ca waves spread throughout the bundles, were closely associated with contraction and depended on L-type Ca channel entry. Immunohistochemistry and Western blotting demonstrated L-type Ca channels, Ca dependent K channels, sarcoplasmic reticulum Ca-adenosine triphosphatase isoforms 2 and 3, inositol triphosphate, and ryanodine receptors. Modulation of Ca and K channel activity was a potent mechanism for affecting Ca and force, whereas manipulation of the sarcoplasmic reticulum had little effect. CONCLUSIONS: To our knowledge this study represents the first measurements of Ca signals in the human ureter obtained during phasic contractions and in response to agonists. Results show that it is controlled by fast propagating Ca waves, which spread rapidly between the muscle bundles, producing regular contractions, and drugs that interfere with excitability or Ca entry through L-type Ca channels have profound effects on Ca signaling and contractility. These data are discussed in relation to the treatment of patients with suspected ureteral dysfunction using Ca entry blockers.

A subset of mucosa-associated Escherichia coli isolates from patients with colon cancer, but not Crohn's disease, share pathogenicity islands with urinary pathogenic E. coli.

Adherent and invasive mucosa-associated Escherichia coli have been implicated in the pathogenesis of colon cancer and inflammatory bowel diseases. It has been reported that such isolates share features of extraintestinal E. coli (ExPEC) and particularly uropathogenic E. coli (UPEC). We used suppression subtractive hybridization (SSH) to subtract the genome of E. coli K-12 from that of a colon cancer mucosal E. coli isolate. Of the subtracted sequences, 53 % were present in the genomes of one or more of three sequenced UPEC strains but absent from the genome of an enterohaemorrhagic E. coli (EHEC) strain. Of the subtracted sequences, 80 % matched at least one UPEC genome, whereas only 4 % were absent from the UPEC genomes but present in the genome of the EHEC strain. A further genomic subtraction against the UPEC strain 536 enriched for sequences matching mobile genetic elements, other ExPEC strains, and other UPEC strains or commensals, rather than strains associated with gastrointestinal disease. We analysed the distribution of selected subtracted sequences and UPEC-associated pathogenicity islands (PAIs) amongst a panel of mucosa-associated E. coli isolated from colonoscopic biopsies of patients with colon cancer, patients with Crohn's disease and controls. This enabled us to identify a group of isolates from colon cancer (30-40 %) carrying multiple genes previously categorized as UPEC-specific and implicated in virulence.

Characterization of epithelial IL-8 response to inflammatory bowel disease mucosal E. coli and its inhibition by mesalamine.

BACKGROUND: Mucosally adherent E. coli are found in inflammatory bowel disease (IBD) and colon cancer. They promote release of the proinflammatory cytokine interleukin-8 (IL-8). We explored mechanisms for this release and its inhibition by drugs. METHODS: IL-8 release from colon epithelial cells in response to mucosal E. coli isolates from IBD, colon cancer, and controls was characterized at the cellular and molecular level. RESULTS: IL-8 response of HT29 cells was greater with Crohn's disease (689 +/- 298 [mean +/- SD] pg IL-8/mL at 4 hours, n = 7) and colon cancer isolates (532 +/- 415 pg/mL, n = 14) than with ulcerative colitis (236 +/- 58 pg/mL, n = 6) or control isolates (236 +/- 100 pg/mL, n = 6, P < 0.0001). Bacterial supernatants contained shed flagellin that triggered IL-8 release. For whole bacteria the IL-8 response to E. coli that agglutinate red blood cells (548 +/- 428 pg IL-8/mL, n = 16), a function that correlates with epithelial invasion, was greater than for nonhemagglutinators (281 +/- 253 pg/mL, n = 17; P < 0.0001). This was particularly marked among E. coli that, although flagellate, could not release IL-8 from TLR5-transfected HEK293 cells. IL-8 release was mediated by extracellular-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) and inhibited by mesalamine, but not hydrocortisone, at therapeutic concentrations. CONCLUSIONS: Mucosa-associated E. coli shed flagellin that elicits epithelial IL-8 release but this may only become relevant when the mucosal barrier is weakened to expose basolateral TLR5. Adherent and invasive IBD and colon cancer E. coli isolates also elicit a flagellin-independent IL-8 response that may be relevant when the mucosal barrier is intact. The IL-8 release is MAPK-dependent and inhibited by mesalamine.

Molecular epidemiology and disease severity of respiratory syncytial virus in relation to other potential pathogens in children hospitalized with acute respiratory infection in Jordan.

Human respiratory syncytial virus (HRSV) is the major viral cause of acute lower respiratory tract infections in children. Few data about the molecular epidemiology of respiratory syncytial virus in developing countries, such as Jordan, are available. The frequency and severity of infections caused by HRSV were assessed in hospitalized Jordanian children <5 years of age compared with other potential etiological agents. Overall a potential pathogen was detected in 78% (254/326) of the children. HRSV was detected in 43% (140/326) of the nasopharyngeal aspirates. HRSV was found more frequently during the winter (January/February), being less frequent or negligible by spring (March/April). Analysis of 135 HRSV-positive strains using restriction fragment length polymorphism showed that 94 (70%) belonged to subgroup A, and 41 (30%) to subgroup B. There were also two cases of mixed genotypic infection. Only four of the six previously described N genotypes were detected with NP4 predominating. There were no associations between subgroup or N-genogroup and disease severity. HRSV was significantly associated with more severe acute respiratory infection and the median age of children with HRSV was lower than for those without. Next in order of frequency were adenovirus (116/312: 37%), human bocavirus (57/312: 18%), rhinovirus (36/325: 11%), Chlamydia spp. (14/312: 4.5%), human metapneumovirus (8/326: 2.5%), human coronavirus NL63 (4/325: 1.2%), and influenza A virus (2/323: 0.6%). Influenza B; parainfluenza viruses 1-4, human coronavirus HKU1 and Mycoplasma pneumoniae were not detected.

Replication of Colonic Crohn's Disease Mucosal Escherichia coli Isolates within Macrophages and Their Susceptibility to Antibiotics.

There is increasing evidence that Escherichia coli organisms are important in Crohn's disease (CD) pathogenesis. In CD tissue they are found within macrophages, and the adherent-invasive CD ileal E. coli isolate LF82 can replicate inside macrophage phagolysosomes. This study investigates replication and antibiotic susceptibility of CD colonic E. coli isolates inside macrophages. Replication of CD colonic E. coli within J774-A1 murine macrophages and human monocyte-derived macrophages (HMDM) was assessed by culture and lysis after gentamicin killing of noninternalized bacteria and verified by electron microscopy (EM). All seven CD colonic isolates tested replicated within J774-A1 macrophages by 3 h (6.36-fold +/- 0.7-fold increase; n = 7 isolates) to a similar extent to CD ileal E. coli LF82 (6.8-fold +/- 0.8-fold) but significantly more than control patient isolates (5.2-fold +/- 0.25-fold; n = 6; P = 0.006) and E. coli K-12 (1.0-fold +/- 0.1-fold; P < 0.0001). Replication of CD E. coli HM605 within HMDM (3.9-fold +/- 0.7-fold) exceeded that for K-12 (1.4-fold +/- 0.2-fold; P = 0.03). EM showed replicating E. coli within macrophage vacuoles. Killing of HM605 within J774-A1 macrophages following a 3-h incubation with antibiotics at published peak serum concentrations (C(max)) was as follows: for ciprofloxacin, 99.5% +/- 0.2%; rifampin, 85.1% +/- 6.6%; tetracycline, 62.8% +/- 6.1%; clarithromycin, 62.1% +/- 5.6% (all P < 0.0001); sulfamethoxazole, 61.3% +/- 7.0% (P = 0.0007); trimethoprim, 56.3% +/- 3.4% (P < 0.0001); and azithromycin, 41.0% +/- 10.5% (P = 0.03). Ampicillin was not effective against intracellular E. coli. Triple antibiotic combinations were assessed at 10% C(max), with ciprofloxacin, tetracycline, and trimethoprim causing 97% +/- 0.0% killing versus 86% +/- 2.0% for ciprofloxacin alone. Colonic mucosa-associated E. coli, particularly CD isolates, replicate within macrophages. Clinical trials are indicated to assess the efficacy of a combination antibiotic therapy targeting intramacrophage E. coli.

Diagnostic multiplex PCR assay for the identification of the Liverpool, Midlands 1 and Manchester CF epidemic strains of Pseudomonas aeruginosa.

Individual PCR amplification tests have been developed for three UK CF epidemic strains, the Liverpool epidemic strain (LES), Midlands 1 and the Manchester epidemic strain (MES). We report a simple diagnostic multiplex PCR test that can be used to screen for all three of these strains. To evaluate the test, we screened collections of LES, MES and Midlands 1 isolates, along with various CF and non-CF non-epidemic Pseudomonas aeruginosa strains. The test was 100% sensitive and 100% specific in the identification of these UK CF epidemic strains.

Microbial mannan inhibits bacterial killing by macrophages: a possible pathogenic mechanism for Crohn's disease.

BACKGROUND & AIMS: Crohn's disease (CD) is mimicked by inherited phagocyte disorders and is associated with circulating antibodies against yeast mannan (anti-Saccharomyces cerevisiae antibody; ASCA). We speculated that mannans might impair phagocyte function. METHODS: S cerevisiae mannan was assessed for its effects on human peripheral blood neutrophils, adherent monocytes, and monocyte-derived macrophages (MDM). RESULTS: Mannan caused dose-related increased survival of CD Escherichia coli HM605 within adherent monocytes from 24% +/- 10.5% (control) to 114% +/- 22.7% with mannan 1 mg/mL at 2 hours (mean +/- SEM, n = 9; P = .0002). Electron microscopy showed E coli HM605 surviving and probably replicating within macrophage vesicles. Mannan (1 mg/mL) inhibited the respiratory burst in neutrophils and monocytes (both P = .002) and bacterial killing within MDM (P < .001). E coli survival was increased within macrophages from TLR4(-/-) (126% +/- 3.5% survival at 2 hours) and MyD88(-/-) (134.8% +/- 6.5%) mice compared with wild-type mice (both P < .0001). Mannan had no additional effect, showing that TLR4 and MyD88 are involved in bacterial killing by macrophages and its inhibition by mannan. Putative CD-associated micro-organisms were screened for the ASCA mannan epitope by Galanthus nivalis lectin (GNA) blotting. ASCA epitope was expressed by Candida albicans and Mycobacterium paratuberculosis but not by Mycobacterium tuberculosis or E coli. Supernatants from M paratuberculosis culture inhibited killing of E coli HM605 by adherent human monocytes and murine macrophages. The inhibitory activity was removed by GNA-affinity chromatography. CONCLUSIONS: Suppression of mucosal phagocyte function by microbial mannans, possibly of Mycobacterial origin, may contribute to CD pathogenesis.

Successful downstream application of the Paxgene Blood RNA system from small blood samples in paediatric patients for quantitative PCR analysis.

BACKGROUND: The challenge of gene expression studies is to reliably quantify levels of transcripts, but this is hindered by a number of factors including sample availability, handling and storage. The PAXgene Blood RNA System includes a stabilizing additive in a plastic evacuated tube, but requires 2.5 mL blood, which makes routine implementation impractical for paediatric use. The aim of this study was to modify the PAXgene Blood RNA System kit protocol for application to small, sick children, without compromising RNA integrity, and subsequently to perform quantitative analysis of ICAM and interleukin-6 gene expression.Aliquots of 0.86 mL PAXgene reagent were put into microtubes and 0.3 mL whole blood added to maintain the same recommended proportions as in the PAXgene evacuated tube system. RNA quality was assessed using the Agilent BioAnalyser 2100 and an in-house TaqMan assay which measures GAPDH transcript integrity by determining 3' to 5' ratios. qPCR analysis was performed on an additional panel of 7 housekeeping genes. Three reference genes (HPRT1, YWHAZ and GAPDH) were identified using the GeNORM algorithm, which were subsequently used to normalising target gene expression levels. ICAM-1 and IL-6 gene expression were measured in 87 Malawian children with invasive pneumococcal disease. RESULTS: Total RNA yield was between 1,114 and 2,950 ng and the BioAnalyser 2100 demonstrated discernible 18s and 28s bands. The cycle threshold values obtained for the seven housekeeping genes were between 15 and 30 and showed good consistency. Median relative ICAM and IL-6 gene expression were significantly reduced in non-survivors compared to survivors (ICAM: 3.56 vs 4.41, p = 0.04, and IL-6: 2.16 vs 6.73, p = 0.02). CONCLUSION: We have successfully modified the PAXgene blood collection system for use in small children and demonstrated preservation of RNA integrity and successful quantitative real-time PCR analysis.

Transcriptomic analysis of the sulfate starvation response of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic pathogen that causes a number of infections in humans, but is best known for its association with cystic fibrosis. It is able to use a wide range of sulfur compounds as sources of sulfur for growth. Gene expression in response to changes in sulfur supply was studied in P. aeruginosa E601, a cystic fibrosis isolate that displays mucin sulfatase activity, and in P. aeruginosa PAO1. A large family of genes was found to be upregulated by sulfate limitation in both isolates, encoding sulfatases and sulfonatases, transport systems, oxidative stress proteins, and a sulfate-regulated TonB/ExbBD complex. These genes were localized in five distinct islands on the genome and encoded proteins with a significantly reduced content of cysteine and methionine. Growth of P. aeruginosa E601 with mucin as the sulfur source led not only to a sulfate starvation response but also to induction of genes involved with type III secretion systems.

High pneumococcal DNA loads are associated with mortality in Malawian children with invasive pneumococcal disease.

BACKGROUND: In bacteremia owing to Streptococcus pneumoniae, high bacterial counts at presentation have been shown to be predictive of the development of serious invasive disease. Using real-time PCR, we aimed to determine pneumococcal DNA loads in blood and CSF, and their relationship to cytokine concentrations, clinical presentation and outcome. METHODS: Children with confirmed meningitis (n = 82) or pneumonia (n = 13) were prospectively recruited, and blood and CSF samples taken for pneumococcal bacterial DNA loads and cytokine determination. RESULTS: At the time of admission, the median bacterial load in blood was 1.6 x 10 DNA copies/mL (range 0.00-1.54 x 10) and in CSF it was 5.77 x 10 DNA copies/mL (range 4.42 x 10 to 6.15 x 10). Median blood and CSF bacterial loads (log DNA copies/mL) were significantly higher in nonsurvivors than in survivors; blood (3.80 vs. 2.97, P = 0.003), CSF (8.17 vs. 7.50, P = 0.03). In HIV-infected children (n = 59), blood and CSF loads and plasma tumor necrosis factor-alpha, interleukin-1beta (IL-1beta), IL-6 and IL-10 were all significantly higher in nonsurvivors than in survivors, but in HIV-uninfected children (n = 36) this difference was not significant. Blood bacterial loads and plasma cytokine concentrations were significantly associated, and were all significantly higher in children with meningitis than in those with pneumonia. In children with meningitis, median CSF cytokine concentrations were significantly higher than median plasma cytokine concentrations (P < 0.001) and CSF bacterial loads were significantly associated with CSF IL-1beta (P = 0.002) and IL-10 (P = 0.001) concentrations. CONCLUSIONS: Pneumococcal DNA loads are associated with plasma cytokine concentrations, and are higher in meningitis than in pneumonia. High blood and CSF pneumococcal DNA loads are associated with a fatal outcome.

Prevalence of feline chronic gingivo-stomatitis in first opinion veterinary practice.

Feline chronic gingivo-stomatitis (FCGS) is a syndrome characterised by persistent, often severe, inflammation of the oral mucosa. In the absence of similar studies, our objective was to estimate the prevalence of FCGS in a convenience based sample of cats visiting first opinion small animal veterinary practices. Twelve practices took part, providing a sample population of 4858 cats. Veterinary surgeons identified cases of FCGS according to our case definition over a 12-week sampling period; age, sex and breed information was determined for all cats, plus brief descriptive data for FCGS cases. The prevalence of FCGS was 0.7% (34 cases, 95% confidence intervals: 0.5-1.0%). Of the 34 cases of FCGS, 44% (15 cats) were new cases and 56% (19 cats) were ongoing cases. No statistically significant difference (P>0.353) was found when the age, sex and breed of cats with FCGS were compared to data from cats without the condition.

Human bocavirus in Iranian children with acute respiratory infections.

Human bocavirus (HBoV), a virus discovered in Sweden in 2005, has been associated with acute respiratory infections in young children and subsequent reports suggest that HBoV may have a worldwide distribution. This report describes the frequency and clinical presentation of HBoV in 261 Iranian children<5 years old with acute respiratory infections attending two regional hospitals in Rasht, Iran in the winter of 2003-2004. Polymerase chain reaction (PCR) and reverse transcription PCR (RT-PCR) were used for the detection of HBoV and other respiratory pathogens from nasopharyngeal specimens. HBoV was detected in 21 (8%) children. Fifteen (12%) of these children were identified among 122 children admitted to hospital and 6 (4%) from 139 outpatients (P < 0.05). Most children with HBoV were less than 2 years (17/21, 81%) and 7 (33%) were less than 1 year old. Although HBoV was identified in all ages it affected slightly older children than the respiratory syncytial virus (RSV). The frequency of the virus varied from 1 (3%) in 40 patients in November to 7 (12%) of 61 in February, suggesting a seasonal pattern during the autumn and early winter. Seven children had co-infections with RSV, adenovirus or influenza A. The relatively high frequency of HBoV suggests that the virus may contribute substantially to acute respiratory infections in children.

Development of a diagnostic test for the Midlands 1 cystic fibrosis epidemic strain of Pseudomonas aeruginosa.

In a previous study of isolates from cystic fibrosis (CF) patients in England and Wales, the Midlands 1 strain of Pseudomonas aeruginosa was identified as the second most common clone, representing 10% of isolates and found in nearly one-third of all CF centres [Scott, F. W. & Pitt, T. L. (2004). J Med Microbiol 53, 609-615]. Using suppression subtractive hybridization, 54 sequences were identified as present in a Midlands 1 strain but were absent from strain PAO1. The distribution of 14 of these sequences amongst representatives of Midlands 1, other CF epidemic strains and unrelated P. aeruginosa CF isolates was determined using PCR assays. Using these data, a PCR-based test was developed that was specific for the Midlands 1 clone, which was confirmed using dot-blot hybridization. By applying the test to CF isolates from a CF centre in Liverpool, a Midlands 1 clone was identified. The identity was confirmed using typing by PFGE. The PCR test should facilitate a greater understanding of the distribution of the Midlands 1 strain in the UK and elsewhere.

Use of suppression subtractive hybridization to examine the accessory genome of the Liverpool cystic fibrosis epidemic strain of Pseudomonas aeruginosa.

The Liverpool epidemic strain (LES) of Pseudomonas aeruginosa has been highly successful at colonizing cystic fibrosis (CF) patients throughout the UK, has replaced previously established strains in CF patients, has caused infections of non-CF parents of CF patients, and can cause greater morbidity in CF than other strains of P. aeruginosa. Using suppression subtractive hybridization (SSH) to identify strain-specific sequences, a diagnostic test for the LES based on PCR amplification of SSH sequence PS21 had previously been developed. In this study, the SSH sequence database of LES was substantially increased, using both extension of previous sequences and new rounds of subtraction. Of 92 SSH sequences identified as present in the LES but absent from strain PAO1, 25 were assessed for prevalence amongst a strain panel consisting mainly of LES and non-LES CF isolates. Preliminary analysis of genome sequence data indicated that all SSH sequences that were LES specific or found only rarely in other strains of P. aeruginosa were present on one of three contigs. All of the SSH sequences screened were either unstable amongst LES isolates or were not completely LES specific. Rare false positives were found with the PS21 test. The authors suggest that a second PCR assay designed to detect SSH sequence LESF9 can be used to confirm the identity of the most prevalent CF epidemic lineage in the UK.