Proposed minimal standards for the description of genera, species and subspecies of the Pasteurellaceae

Principles and guidelines are presented to ensure a solid scientific standard of papers dealing with the taxonomy of taxa of Pasteurellaceae Pohl 1981. The classification of the Pasteurellaceae is in principle based on a polyphasic approach. DNA sequencing of certain genes is very important for defining the borders of a taxon. However, the characteristics that are common to all members of the taxon and which might be helpful for separating it from related taxa must also be identified. Descriptions have to be based on as many strains as possible (inclusion of at least five strains is highly desirable), representing different sources with respect to geography and ecology, to allow proper characterization both phenotypically and genotypically, to establish the extent of diversity of the cluster to be named. A genus must be monophyletic based on 16S rRNA gene sequence-based phylogenetic analysis. Only in very rare cases is it acceptable that monophyly can not be achieved by 16S rRNA gene sequence comparison. Recently, the monophyly of genera has been confirmed by sequence comparison of housekeeping genes. In principle, a new genus should be recognized by a distinct phenotype, and characters that separate the new genus from its neighbours should be given clearly. Due to the overall importance of accurate classification of species, at least two genotypic methods are needed to show coherence and for separation at the species level. The main criterion for the classification of a novel species is that it forms a monophyletic group based on 16S rRNA gene sequence-based phylogenetic analysis. However, some groups might also include closely related species. In these cases, more sensitive tools for genetic recognition of species should be applied, such as DNA-DNA hybridizations. The comparison of housekeeping gene sequences has recently been used for genotypic definition of species. In order to separate species, phenotypic characters must also be identified to recognize them, and at least two phenotypic differences from existing species should be identified if possible. We recommend the use of the subspecies category only for subgroups associated with disease or similar biological characteristics. At the subspecies level, the genotypic groups must always be nested within the boundaries of an existing species. Phenotypic cohesion must be documented at the subspecies level and separation between subspecies and related species must be fully documented, as well as association with particular disease and host. An overview of methods previously used to characterize isolates of the Pasteurellaceae has been given. Genotypic and phenotypic methods are separated in relation to tests for investigating diversity and cohesion and to separate taxa at the level of genus as well as species and subspecies.

A novel secretin receptor splice variant potentially useful for early diagnosis of pancreatic carcinoma

BACKGROUND ; AIMS: Pancreatic and bile duct carcinomas represent highly aggressive malignancies that evolve from secretin receptor-rich ductular cells. With premessenger RNA splicing abnormalities common in cancer, we evaluated whether an abnormal secretin receptor spliceoform were present, characterized it, and developed a serum assay for it. METHODS: Cancer cell lines and healthy and neoplastic tissue were studied by nested reverse-transcription polymerase chain reaction and sequencing. A promising spliceoform was isolated and characterized, and monoclonal antibodies were raised to 2 distinct regions. A dual antibody enzyme-linked immunosorbent assay was developed and applied to blinded serum samples from 26 patients with pancreatic carcinoma, 10 patients with chronic pancreatitis, and 14 controls. RESULTS: Each of 9 pancreatic cancer specimens and no normal tissue expressed a secretin receptor variant with exons 3 and 4 deleted. This encoded a 111-residue peptide with its first 43 residues identical to wild-type receptor, but, subsequent to a shift in coding frame and early truncation, the next 68 residues were unique in the transcriptome/proteome. This nonfunctional soluble protein did not bind or signal in response to secretin and was secreted from transfected MiaPaCa-2 cells. Elevated serum levels of this variant were present in 69% of pancreatic cancer patients, 60% of chronic pancreatitis patients, and 1 of 14 controls. CONCLUSIONS: We identified a novel abnormal spliceoform of the secretin receptor in pancreatic and bile duct cancers and developed a dual antibody sandwich enzyme-linked immunosorbent assay to measure it in the circulation. Initial application of this assay in patients with pancreatic cancer and chronic pancreatitis was promising, but additional validation will be required to evaluate its clinical utility.

Plasma leptin levels in men are not related to the development of lipoatrophy during antiretroviral therapy

OBJECTIVES: To assess the correlations between the hormone leptin and lipoatrophy in HIV-positive, treatment-naive patients on combination antiretroviral therapy (cART). DESIGN: Case-control study nested in a multicentre cohort of HIV-infected adults. Cases were patients that developed lipoatrophy and controls those who did not. PATIENTS AND METHODS: Clinical parameters and plasma leptin determinations were studied in 97 HIV-1-infected, treatment-naive Caucasian men (10 cases and 87 controls) on an unchanged and virologically successful drug regimen with a zidovudine/lamivudine backbone at baseline and after 2 years of cART. The association of plasma leptin levels and the development of lipoatrophy was investigated. RESULTS: Two years of cART was not associated with a change in plasma leptin levels. Plasma leptin levels remained sensible to changes in body mass index. There was no difference in leptin levels between patients who developed lipoatrophy and controls, neither before nor after cART. The only predictor of development of lipoatrophy was a higher age (P = 0.02). CONCLUSIONS: Leptin as measured in plasma is unlikely to play a major role in the genesis of lipoatrophy.

Economic burden of surgical site infections at a European university hospital

OBJECTIVE: To quantify the economic burden of in-hospital surgical site infections (SSIs) at a European university hospital. DESIGN: Matched case-control study nested in a prospective observational cohort study. SETTING: Basel University Hospital in Switzerland, where an average of 28,000 surgical procedures are performed per year. METHODS: All in-hospital occurrences of SSI associated with surgeries performed between January 1, 2000, and December 31, 2001, by the visceral, vascular, and traumatology divisions at Basel University Hospital were prospectively recorded. Each case patient was matched to a control patient by age, procedure code, and National Nosocomial Infection Surveillance System risk index. The case-control pairs were analyzed for differences in cost of hospital care and in provision of specialized care. RESULTS: A total of 6,283 procedures were performed: 187 SSIs were detected in inpatients, 168 of whom were successfully matched with a control patient. For case patients, the mean additional hospital cost was SwF-19,638 (95% confidence interval [CI], SwF-8,492-SwF-30,784); the mean additional postoperative length of hospital stay was 16.8 days (95% CI, 13-20.6 days); and the mean additional in-hospital duration of antibiotic therapy was 7.4 days (95% CI, 5.1-9.6 days). Differences were primarily attributable to organ space SSIs (n = 76). CONCLUSIONS: In a European university hospital setting, SSIs are costly and constitute a heavy and potentially preventable burden on both patients and healthcare providers.

Influence of HIV-related immunodeficiency on the risk of hepatocellular carcinoma

OBJECTIVE: To investigate HIV-related immunodeficiency as a risk factor for hepatocellular carcinoma (HCC) among persons infected with HIV, while controlling for the effect of frequent coinfection with hepatitis C and B viruses. DESIGN: A case-control study nested in the Swiss HIV Cohort Study. METHODS: Twenty-six HCC patients were identified in the Swiss HIV Cohort Study or through linkage with Swiss Cancer Registries, and were individually matched to 251 controls according to Swiss HIV Cohort Study centre, sex, HIV-transmission category, age and year at enrollment. Odds ratios and corresponding confidence intervals were estimated by conditional logistic regression. RESULTS: All HCC patients were positive for hepatitis B surface antigen or antibodies against hepatitis C virus. HCC patients included 14 injection drug users (three positive for hepatitis B surface antigen and 13 for antibodies against hepatitis C virus) and 12 men having sex with men/heterosexual/other (11 positive for hepatitis B surface antigen, three for antibodies against hepatitis C virus), revealing a strong relationship between HIV transmission route and hepatitis viral type. Latest CD4+ cell count [Odds ratio (OR) per 100 cells/mul decrease = 1.33, 95% confidence interval (CI) 1.06-1.68] and CD4+ cell count percentage (OR per 10% decrease = 1.65, 95% CI 1.01-2.71) were significantly associated with HCC. The effects of CD4+ cell count were concentrated among men having sex with men/heterosexual/other rather than injecting drug users. Highly active antiretroviral therapy use was not significantly associated with HCC risk (OR for ever versus never = 0.59, 95% confidence interval 0.18-1.91). CONCLUSION: Lower CD4+ cell counts increased the risk for HCC among persons infected with HIV, an effect that was particularly evident for hepatitis B virus-related HCC arising in non-injecting drug users.

Sampling-Design Effects on Properties of Species-Area Relationships - A Case Study from Estonian Dry Grassland Communities

Despite widespread use of species-area relationships (SARs), dispute remains over the most representative SAR model. Using data of small-scale SARs of Estonian dry grassland communities, we address three questions: (1) Which model describes these SARs best when known artifacts are excluded? (2) How do deviating sampling procedures (marginal instead of central position of the smaller plots in relation to the largest plot; single values instead of average values; randomly located subplots instead of nested subplots) influence the properties of the SARs? (3) Are those effects likely to bias the selection of the best model? Our general dataset consisted of 16 series of nested-plots (1 cm(2)-100 m(2), any-part system), each of which comprised five series of subplots located in the four corners and the centre of the 100-m(2) plot. Data for the three pairs of compared sampling designs were generated from this dataset by subsampling. Five function types (power, quadratic power, logarithmic, Michaelis-Menten, Lomolino) were fitted with non-linear regression. In some of the communities, we found extremely high species densities (including bryophytes and lichens), namely up to eight species in 1 cm(2) and up to 140 species in 100 m(2), which appear to be the highest documented values on these scales. For SARs constructed from nested-plot average-value data, the regular power function generally was the best model, closely followed by the quadratic power function, while the logarithmic and Michaelis-Menten functions performed poorly throughout. However, the relative fit of the latter two models increased significantly relative to the respective best model when the single-value or random-sampling method was applied, however, the power function normally remained far superior. These results confirm the hypothesis that both single-value and random-sampling approaches cause artifacts by increasing stochasticity in the data, which can lead to the selection of inappropriate models.

Hodgkin lymphoma in the Swiss HIV Cohort Study

Hodgkin lymphoma (HL) risk is elevated among persons infected with HIV (PHIV) and has been suggested to have increased in the era of combined antiretroviral therapy (cART). Among 14,606 PHIV followed more than 20 years in the Swiss HIV Cohort Study (SHCS), determinants of HL were investigated using 2 different approaches, namely, a cohort and nested case-control study, estimating hazard ratios (HRs) and matched odds ratios, respectively. Forty-seven incident HL cases occurred during 84,611 person-years of SHCS follow-up. HL risk was significantly higher among men having sex with men (HR vs intravenous drug users = 2.44, 95% confidence interval [CI], 1.13-5.24) but did not vary by calendar period (HR for 2002-2007 vs 1995 or earlier = 0.65, 95% CI, 0.29-1.44) or cART use (HR vs nonusers = 1.02, 95% CI, 0.53-1.94). HL risk tended to increase with declining CD4(+) cell counts, but these differences were not significant. A lower CD4(+)/CD8(+) ratio at SHCS enrollment or 1 to 2 years before HL diagnosis, however, was significantly associated with increased HL risk. In conclusion, HL risk does not appear to be increasing in recent years or among PHIV using cART in Switzerland, and there was no evidence that HL risk should be increased in the setting of improved immunity.

JC virus-specific immune responses in human immunodeficiency virus type 1 patients with progressive multifocal leukoencephalopathy

Progressive multifocal leukoencephalopathy (PML) is a frequently fatal disease caused by uncontrolled polyomavirus JC (JCV) in severely immunodeficient patients. We investigated the JCV-specific cellular and humoral immunity in the Swiss HIV Cohort Study. We identified PML cases (n = 29), as well as three matched controls per case (n = 87), with prospectively cryopreserved peripheral blood mononuclear cells and plasma at diagnosis. Nested controls were matched according to age, gender, CD4(+) T-cell count, and decline. Survivors (n = 18) were defined as being alive for >1 year after diagnosis. Using gamma interferon enzyme-linked immunospot assays, we found that JCV-specific T-cell responses were lower in nonsurvivors than in their matched controls (P = 0.08), which was highly significant for laboratory- and histologically confirmed PML cases (P = 0.004). No difference was found between PML survivors and controls or for cytomegalovirus-specific T-cell responses. PML survivors showed significant increases in JCV-specific T cells (P = 0.04) and immunoglobulin G (IgG) responses (P = 0.005). IgG responses in survivors were positively correlated with CD4(+) T-cell counts (P = 0.049) and negatively with human immunodeficiency virus RNA loads (P = 0.03). We conclude that PML nonsurvivors had selectively impaired JCV-specific T-cell responses compared to CD4(+) T-cell-matched controls and failed to mount JCV-specific antibody responses. JCV-specific T-cell and IgG responses may serve as prognostic markers for patients at risk.

Assessment of a real-time PCR test to diagnose syphilis from diverse biological samples

OBJECTIVES: To investigate the contribution of a real-time PCR assay for the detection of Treponema pallidum in various biological specimens with the secondary objective of comparing its value according to HIV status. METHODS: Prospective cohort of incident syphilis cases from three Swiss hospitals (Geneva and Bern University Hospitals, Outpatient Clinic for Dermatology of Triemli, Zurich) diagnosed between January 2006 and September 2008. A case-control study was nested into the cohort. Biological specimens (blood, lesion swab or urine) were taken at diagnosis (as clinical information) and analysed by real-time PCR using the T pallidum 47 kDa gene. RESULTS: 126 specimens were collected from 74 patients with primary (n = 26), secondary (n = 40) and latent (n = 8) syphilis. Among primary syphilis, sensitivity was 80% in lesion swabs, 28% in whole blood, 55% in serum and 29% in urine, whereas among secondary syphilis, it was 20%, 36%, 47% and 44%, respectively. Among secondary syphilis, plasma and cerebrospinal fluid were also tested and provided a sensitivity of 100% and 50%, respectively. The global sensitivity of T pallidum by PCR (irrespective of the compartment tested) was 65% during primary, 53% during secondary and null during latent syphilis. No difference regarding serology or PCR results was observed among HIV-infected patients. Specificity was 100%. CONCLUSIONS: Syphilis PCR provides better sensitivity in lesion swabs from primary syphilis and displays only moderate sensitivity in blood from primary and secondary syphilis. HIV status did not modify the internal validity of PCR for the diagnosis of primary or secondary syphilis.

Color polymorphism and intrasexual competition in assemblages of cichlid fish

The origin and maintenance of phenotypic polymorphisms is a classical problem in evolutionary ecology. Aggressive male-male competition can be a source of negative frequency-dependent selection stabilizing phenotypic polymorphisms when aggression is biased toward the own morph. We studied experimental assemblages of red and blue color morphs of the Lake Victoria cichlid fish Pundamilia. Aggression was investigated in mixed-color and single-color assemblages. We found that aggression was indeed biased toward males of the same color, which could in theory reduce aggression levels in mixed-color assemblages and promote coexistence. However, previous studies showed high aggression levels in red and dominance of red over blue males in dyadic interactions, which could hinder coexistence. We found that coexistence in mixed-color assemblages reduced the level of aggression in red males but not in blue males. Red and blue males were equally dominant in mixed-color assemblages, suggesting that predictions derived from dyadic interactions may not be valid for an assemblage situation. The results are consistent with field data: the geographic range of red is nested within that of blue, suggesting that red cannot displace blue. Our study suggests that male-male competition may be a significant force for maintaining phenotypic diversity.

Dry grasslands of NW Bulgarian mountains: first insights into diversity, ecology and syntaxonomy

We present the data of the 3rdresearch expedition of the European Dry Grasslands Group (EDGG), which was conducted in 2011 in two contrasting areas in NW Bulgarian mountains. The aim was to collect plot data for comparing Bulgarian dry grasslands with those of other parts of Europe in terms of syntaxonomy and biodiversity. We sampled 15 nested-plot series (0.0001–100 m²) and 68 normal plots(10 m²) covering the full variety of dry grassland types occurring in the Vratsa area (Balkan Mts.) and the Koprivshtitsa area (Sredna Gora Mt.). In the plots all vascular plants, terricolous non-vascular plants and a set of soil and other environmental parameters were determined. By applying modified TWIN-SPAN, we distinguished 10 floristically well characterised vegetation types at the association level. After comparison with the regional and European literature, we propose to place them within three classes and five orders: Festuco-Brometea with the orders Stipo pulcherrimae-Festucetalia pallentis (xerophilous dry grasslands of base-rich rocks; alliance Saturejion montanae), Brachypodietalia pinnate (meso-xeric, basiphilous grasslands; alliances Cirsio-Brachypodion pinnate and Chyrsopogono grylli-Danthonion calycinae),Calluno-Ulicetea with the order Nardetalia stricae (lowland to montane Nardus swards; alliance Violion caninae), and Koelerio-Corynephoretea with the orders Sedo-Scleranthetalia (open communities of skeleton-rich, acidic soils; alliance unclear) and Trifolioarvensis-Festucetalia ovinae(closed, meso-xeric, acidophilous grasslands; alliance Armerio rumelicae-Potentillion). The Violion caninae with the association Festuco rubrae-Genistelletum sagittalisis reported from Bulgaria for the first time, while the two occurring Koelerio-Corynephoretea communities are described as new associations (Cetrario aculeatae-Plantaginetum radicatae, Plantagini radicatae-Agrostietum capillaris). According to DCA the main floristic gradient was largely determined by soil conditions, differentiating the Festuco-Brometea communities on soils with high pH and high humus content from the Koelerio-Corynephoretea communities on acidic, humus-poor soils, while the Calluno-Ulicetea stands are the connecting link. At 10 m² Festuco-Brometea and Calluno-Ulicetea stands were richer in species across all investigated taxa and in vascular plants than Koelerio-Corynephoretea stands; the latter were richest in lichen species, while bryophyte richness did not differ significantly among syntaxa. Among the Bulgarian classes, the species-area relationships tended to be steepest in the Festuco-Brometea (i.e. highest beta diversity), but both alpha and beta diversity clearly fell behind the Festuco-Brometea communities in the Transylvanian Plateau, Romania, located less than 500 km north of the study region. Overall, our study contributes to a more adequate placement of the Bulgarian dry grasslands in the European syntaxonomic system and provides valuable data for large-scale analyses of biodiversity patterns

Identification and detection of Actinobacillus pleuropneumoniae by PCR based on the gene apxIVA

The apxIVA gene, a recently discovered RTX determinant of Actinobacillus pleuropneumoniae, was shown to be species-specific. DNA hybridization experiments using probes for various regions of apxIVA revealed that the 3'-terminus of this gene was present in all 14 serotypes of A. pleuropneumoniae but absent from phylogenetically related species. A primer pair spanning this region specifically amplified a 422bp fragment in PCR experiments with DNA from the reference strains of the 14 serotypes and 194 field strains isolated from various geographic locations worldwide. DNA sequence analysis of PCR products derived from all serotypes were identical except in serotypes 3, 8, and 10, which showed minor differences. The PCR did not amplify any product when DNA from 17 different bacterial species closely related to A. pleuropneumoniae was used as template. In addition, the PCR was negative with DNA of several Actinobacillus sp. which were initially characterized as A. pleuropneumoniae using routine phenotypic and serological analyses but which were subsequently shown by 16S rRNA sequence analysis to belong to yet undefined Actinobacillus species. The sensitivity of the PCR was determined to be 10pg of A. pleuropneumoniae DNA. A set of nested primers amplified a 377bp fragment specifically with A. pleuropneumoniae DNA. DNA titration experiments using the flanking and nested primer pairs showed an improved level of sensitivity to approximately 10fg of genomic DNA. The nested PCR was used to monitor the spread of A. pleuropneumoniae in pigs experimentally infected with a virulent serotype 1 strain and housed in a controlled environment facility. A. pleuropneumoniae DNA could be detected by nested PCR in nasal swab samples of infected pigs receiving either a high dose (5x10(5)) or a low dose (1x10(4)) challenge and in unchallenged cohorts that were contact-infected by the inoculated animals. Furthermore, PCR confirmed the presence of A. pleuropneumoniae in 16/17 homogenates from necrotic lung lesions, while the bacterium was successfully recovered from 13 of these lesions by culture.