bluuebunny/crossref_metadata_2025 huggingface dataset:
A retrospective study of surgically treated cases of septic peritonitis in the cat (2000–2007)
Authors: Parsons K. J. , Owen L. J. , Lee K. , Tivers M. S. , Gregory S. P.
Published: October 2009

Objectives:To review aetiology, clinical signs and outcome of cats surgically treated for septic peritonitis (2000‐2007).

Methods:A retrospective study. Inclusion criteria were the identification of intracellular bacteria and degenerate neutrophils and/or a positive culture from abdominal fluid and exploratory coeliotomy. Aetiology, clinical signs, haematological and biochemical parameters, surgical treatment and outcome were recorded and analysed.

Results:Twenty‐six cats fulfilled the inclusion criteria. Abdominal pain was reported in 10 (38 per cent) and vomiting was reported in 11 (42 per cent) of the cats. The most common aetiology was trauma (31 per cent). The principal source of contamination was the gastrointestinal tract. Hyperlactataemia, hypoproteinaemia and hyperglycaemia were reported in 9, 13 and 14 of the 26 cases, respectively. Non‐survivors had significantly higher blood lactate concentrations than survivors (P=0·02). Nineteen cats were managed with primary closure, two with closed suction drains and three with open peritoneal drainage. Twelve (46 per cent) cats survived to discharge.

ClinicalSignificance:In cats, lethargy, depression and anorexia were more common clinical signs than abdominal pain. Lactate level at the time of diagnosis may be a useful prognostic indicator in cats. The proportion of cats that survived was lower than previously reported and owners should be given a guarded prognosis.
The effect of substrate composition and storage time on urine specific gravity in dogs
Authors: Steinberg E. , Drobatz K. , Aronson L.
Published: October 2009

Objectives: The purpose of this study is to evaluate the effects of substrate composition and storage time on urine specific gravity in dogs.

Methods: A descriptive cohort study of 15 dogs. The urine specific gravity of free catch urine samples was analysed during a 5‐hour time period using three separate storage methods; a closed syringe, a diaper pad and non‐absorbable cat litter.

Results: The urine specific gravity increased over time in all three substrates. The syringe sample had the least change from baseline and the diaper sample had the greatest change from baseline. The urine specific gravity for the litter and diaper samples had a statistically significant increase from the 1‐hour to the 5‐hour time point.

Clinical Significance: The urine specific gravity from canine urine stored either on a diaper or in a non‐absorbable litter increased over time. Although the change was found to be statistically significant over the 5‐hour study period it is unlikely to be clinically significant.
The effect of omeprazole on oesophageal pH in dogs during anaesthesia
Authors: Panti A. , Bennett R.C. , Corletto F. , Brearley J. , Jeffrey N. , Mellanby R.J.
Published: October 2009

Objectives:Intra‐operative, gastro‐oesophageal reflux may be associated with post‐anaesthestic complications such as oesophagitis and oesophageal stricture in dogs. The aim of this study was to investigate the effect of preoperative administration of omeprazole, a proton pump inhibitor, on oesophageal pH in anaesthetised dogs.

Methods:Forty‐seven dogs undergoing elective pelvic limb orthopaedic surgery were enrolled into the study. These were randomly allocated to treatment group (n=22) or control group (n=25). The treatment group received one dose of omeprazole (1 mg/kg po) at least 4 h before anaesthesia. All dogs were anaesthetised by the same standardised protocol. A pH probe was inserted into the distal oesophagus after induction of anaesthesia and oesophageal pH was continuously monitored.

Results:In the treatment group, four animals (18 per cent) showed a sudden decrease in oesophageal pH (<4). In the control group the same phenomenon was detected in 13 animals (52 per cent). Gastro‐oesophageal reflux occurred more frequently in the control group compared with the omeprazole group (odds ratio 4·7, 95 per cent C.I. 1.1 to 24.7, P=0.032).

ClinicalSignificance:This study suggests that the preoperative administration of omeprazole is effective in reducing the incidence of gastro‐oesophageal reflux during anaesthesia in dogs.
Parentage Determination and Effective Population Size Estimation in Mass Spawning Pacific Oyster, Crassostrea gigas, Based on Microsatellite Analysis
Authors: Li Ronghua , L Qi , Yu Ruihai
Published: October 2009

Seven high polymorphic microsatellite loci were used to determine the pedigrees in a mass spawning of Pacific oyster, Crassostrea gigas, and to estimate the genetic variability between broodstock and offspring. Parental assignment was performed on a total of 155 individuals, including 141 offspring, 8 candidate mothers, and 6 candidate fathers. The assignment results of real offspring were generally in agreement with simulation with a success rate over 99% using only six of these loci. The allelic diversity and observed heterozygosity (Ho) exhibited similarity between parents and offspring populations, but the expected heterozygosity (He) had a significant decrease in offspring. Although all the males and females contributed to the next generation, the variances of reproductive success and unequal sex ratio resulted in a decline in effective population size (Ne = 11.42). The inbreeding rate of this small‐scale, mass spawning population was estimated at approximately 16.5% per generation. This gave us an insight that when designing breeding programs based on mass spawning for future oyster cultivation generations, the higher inbreeding and lower effective population size should be considered.
Composition, Properties and Sensory Quality of Rainbow Trout Affected by Feed Formulations
Authors: Lesiow Tomasz , Ockerman Herbert W. , Dabrowski Konrad
Published: October 2009

Rainbow trout were fed a fish meal‐based diet or diets where 25, 50, 75, or 100% of the fish meal protein was replaced with a mixture of rendered animal protein ingredients. Diets were formulated to be iso‐nitrogenous (36% crude protein) and meet all known nutrient requirements of rainbow trout. The effect of five diets on dressing percentage, chemical composition, functional properties, and sensory characteristics of rainbow trout fillets stored up to 1 wk on ice in a refrigerated room (4 C) was determined. Dressing percentage based on body weight (from 724 to 811 g) did not show a significant difference due to the diet. Raw tissue pH, 24 and 144 h post mortem, were significantly lower than the control when 75 and 100% fish meal replacement were utilized. Assessment of color, texture, flavor, and tenderness indicated that fillet were lighter, less firm, more bland, and more tender after 144 h compared to 24‐h storage time. Fillets from fish fed all diets were considered as acceptable by the panel.
Dietary Zinc Requirement of Hybrid Striped Bass,Morone chrysops×Morone saxatilis, and Bioavailability of Two Chemically Different Zinc Compounds
Authors: Buentello J. Alejandro , Goff Jonathan B. , Gatlin Delbert M.
Published: October 2009

Zinc is a trace mineral element that plays an essential role in numerous biochemical processes, and has been shown to affect growth and health of several fish species. However, the dietary zinc requirement of hybrid striped bass has not been defined. Therefore, a feeding trial was conducted to determine the dietary requirement for zinc by this fish and to compare zinc bioavailability of two chemically different forms (zinc sulfate and zinc proteinate). Six experimental diets were formulated with purified ingredients and supplemented with ZnSO4to provide total zinc concentrations of 7, 12, 16, 26, 42, and 80 mg/kg diet which were determined by analysis. Each diet contained 32% crude protein, 6% lipid, and approximately 14.2 kJ of digestible energy per gram. The experimental diets were fed twice daily for 10 wk to triplicate groups of 15 hybrid striped bass initially weighing 0.86 ± 0.05 g/fish in 38‐L glass aquaria, connected as a recirculating system. Finally, after the feeding period, the fish were evaluated for weight gain, feed efficiency, protein efficiency ratio, and survival, as well as blood serum zinc, bone zinc, and hematocrit. All fish thrived during the feeding trial and not even the fish fed the basal diet displayed any apparent deficiency signs, although weight gain steadily increased with escalating levels of dietary zinc up to 42 mg/kg diet. On the basis of the most responsive indicators–bone zinc and serum zinc–the minimum dietary zinc requirement of hybrid striped bass was determined to be 17.0 and 17.3 mg Zn/kg diet, respectively, based on broken‐line regression. This estimate is similar in magnitude to dietary zinc requirements reported for other fish species. In addition, the bioavailability of zinc proteinate versus that of ZnSO4was estimated by deriving the ratio of the slopes of the regression lines fitted to bone zinc and serum zinc data. This analysis indicated that hybrid striped bass utilized zinc proteinate ∼1.7 times more efficiently than ZnSO4.
Enhanced Sulfur and Coking Tolerance of a Mixed Ion Conductor for SOFCs: BaZr
0.1
Ce
0.7
Y
0.2–

x

Yb

x

O
3–δ
Authors: Yang Lei , Wang Shizhong , Blinn Kevin , Liu Mingfei , Liu Ze , Cheng Zhe , Liu Meilin
Published: October 2009

Cleaning Solid Oxide Fuel Cells

Solid oxide fuel cells, which operate between 500° and 1000°C, transport oxygen through a ceramic material. At these temperatures, metals that catalyze hydrocarbon reforming reactions can also be incorporated so that conventional fuels such as methane can power the cell. One problem, however, has been rapid deactivation by sulfur impurities and carbon buildup.

Yang
et al.

(p.
126
; see the Perspective by

Selman

) report that doping of a barium zirconate-cerate with the rare-earths Y and Yb creates a material that transports both protons and oxygen ions at 750°C. This material, when used with nickel at the fuel cell anode, resists deactivation even when traces of hydrogen sulfide are present, apparently through enhanced ability to supply or remove water during surface reactions.
Dissecting the Genetic Basis of Resistance to Malaria Parasites in
Anopheles gambiae
Authors: Blandin Stephanie A. , Wang-Sattler Rui , Lamacchia Marina , Gagneur Julien , Lycett Gareth , Ning Ye , Levashina Elena A. , Steinmetz Lars M.
Published: October 2009

Variable Defenses

Recent mapping of resistance alleles in the mosquito
Anopheles gambiae
that provide protection against the human malaria parasite
Plasmodium falciparum
revealed a major
Plasmodium
resistance island (PRI), comprising allelic versions of two leucine-rich repeat-containing proteins, LRIM1 and APL1, which form a complex with the complement C3-like protein TEP1. Using RNA interference inactivation of heterozygous allelic versions of
TEP1
genes,

Blandin
et al.

(p.
147
) show that TEP1 heterogeneity reflects phenotypic variation among mosquito strains parasitized with the rodent malaria parasite
Plasmodium berghei
. It remains unclear whether the observed differences are the outcomes of different selection regimes, because of differing mechanisms, or because the complex is also used in other contexts.
Macrovertebrate Paleontology and the Pliocene Habitat of
Ardipithecus ramidus
Authors: White Tim D. , Ambrose Stanley H. , Suwa Gen , Su Denise F. , DeGusta David , Bernor Raymond L. , Boisserie Jean-Renaud , Brunet Michel , Delson Eric , Frost Stephen , Garcia Nuria , Giaourtsakis Ioannis X. , Haile-Selassie Yohannes , Howell F. Clark , Lehmann Thomas , Likius Andossa , Pehlevan Cesur , Saegusa Haruo , Semprebon Gina , Teaford Mark , Vrba Elisabeth
Published: October 2009

A diverse assemblage of large mammals is spatially and stratigraphically associated with
Ardipithecus ramidus
at Aramis. The most common species are tragelaphine antelope and colobine monkeys. Analyses of their postcranial remains situate them in a closed habitat. Assessment of dental mesowear, microwear, and stable isotopes from these and a wider range of abundant associated larger mammals indicates that the local habitat at Aramis was predominantly woodland. The
Ar. ramidus
enamel isotope values indicate a minimal C
4
vegetation component in its diet (plants using the C
4
photosynthetic pathway), which is consistent with predominantly forest/woodland feeding. Although the Early Pliocene Afar included a range of environments, and the local environment at Aramis and its vicinity ranged from forests to wooded grasslands, the integration of available physical and biological evidence establishes
Ar. ramidus
as a denizen of the closed habitats along this continuum.
Paleobiological Implications of the
Ardipithecus ramidus
Dentition
Authors: Suwa Gen , Kono Reiko T. , Simpson Scott W. , Asfaw Berhane , Lovejoy C. Owen , White Tim D.
Published: October 2009

The Middle Awash
Ardipithecus ramidus
sample comprises over 145 teeth, including associated maxillary and mandibular sets. These help reveal the earliest stages of human evolution.
Ar. ramidus
lacks the postcanine megadontia of
Australopithecus
. Its molars have thinner enamel and are functionally less durable than those of
Australopithecus
but lack the derived
Pan
pattern of thin occlusal enamel associated with ripe-fruit frugivory. The
Ar. ramidus
dental morphology and wear pattern are consistent with a partially terrestrial, omnivorous/frugivorous niche. Analyses show that the
ARA-VP-6/500
skeleton is female and that
Ar. ramidus
was nearly monomorphic in canine size and shape. The canine/lower third premolar complex indicates a reduction of canine size and honing capacity early in hominid evolution, possibly driven by selection targeted on the male upper canine.
The
Ardipithecus ramidus
Skull and Its Implications for Hominid Origins
Authors: Suwa Gen , Asfaw Berhane , Kono Reiko T. , Kubo Daisuke , Lovejoy C. Owen , White Tim D.
Published: October 2009

The highly fragmented and distorted skull of the adult skeleton
ARA-VP-6/500
includes most of the dentition and preserves substantial parts of the face, vault, and base. Anatomical comparisons and micro–computed tomography–based analysis of this and other remains reveal pre-
Australopithecus
hominid craniofacial morphology and structure. The
Ardipithecus ramidus
skull exhibits a small endocranial capacity (300 to 350 cubic centimeters), small cranial size relative to body size, considerable midfacial projection, and a lack of modern African ape–like extreme lower facial prognathism. Its short posterior cranial base differs from that of both
Pan troglodytes
and
P. paniscus
.
Ar. ramidus
lacks the broad, anteriorly situated zygomaxillary facial skeleton developed in later
Australopithecus
. This combination of features is apparently shared by
Sahelanthropus
, showing that the Mio-Pliocene hominid cranium differed substantially from those of both extant apes and
Australopithecus
.
Global Surface Wave Tomography Using Seismic Hum
Authors: Nishida Kiwamu , Montagner Jean-Paul , Kawakatsu Hitoshi
Published: October 2009

Long-period surface waves from oceanic or atmospheric disturbances can be used for seismic mapping of the upper mantle.
Ribosomal Protein S6 Kinase 1 Signaling Regulates Mammalian Life Span
Authors: Selman Colin , Tullet Jennifer M. A. , Wieser Daniela , Irvine Elaine , Lingard Steven J. , Choudhury Agharul I. , Claret Marc , Al-Qassab Hind , Carmignac Danielle , Ramadani Faruk , Woods Angela , Robinson Iain C. A. , Schuster Eugene , Batterham Rachel L. , Kozma Sara C. , Thomas George , Carling David , Okkenhaug Klaus , Thornton Janet M. , Partridge Linda , Gems David , Withers Dominic J.
Published: October 2009

Mimicking Caloric Restriction

The extended life span and resistance to age-related diseases in animals exposed to caloric restriction has focused attention on the biochemical mechanisms that produce these effects.

Selman
et al.

(p.
140
; see the Perspective by

Kaeberlein and Kapahi
)

explored the role of the mammalian ribosomal protein S6 kinase 1 (S6K1), which regulates protein translation and cellular energy metabolism. Female knockout mice lacking expression of S6K1 showed characteristics of animals exposed to caloric restriction, including improved health and increased longevity. The beneficial effects included reduced fat mass in spite of increased food intake. Thus, inhibition of signaling pathways activated by S6K1 might prove beneficial in protecting against age-related disease.
The Coordinates of Truth
Authors: Nabel Gary J.
Published: October 2009

What drives scientific discovery, and how can we make it happen more effectively?
Nitrous Oxide: No Laughing Matter
Authors: Wuebbles Donald J.
Published: October 2009

Rising atmospheric concentrations of nitrous oxide are contributing to global warming and stratospheric ozone destruction.
A Revolving Door for Calcium
Authors: Demaurex Nicolas , Poburko Damon
Published: October 2009

A mitochondrial protein linked to seizures exchanges calcium and hydrogen ions, controlling cell respiration and calcium signaling.
Mars Mission Delayed as Mad Dash to Prep Probe Falls Short
Authors: Stone Richard
Published: October 2009

Russia and China postpone until 2011 a mission to send a pair of probes to Mars and Phobos, the larger of the two martian moons, and return samples from Phobos—the first such mission in more than 30 years.
Scientists Decry Isotope, DNA Testing of ‘Nationality’
Authors: Travis John
Published: October 2009

Scientists are greeting with dismay a project to use DNA and isotope analysis of tissue from asylum-seekers to evaluate their nationality and help decide who can enter the United Kingdom.
On the Origin of Ecological Structure
Authors: Stokstad Erik
Published: October 2009

What dictates the kinds and proportions of organisms in a community? In the 10th essay in
Science
's series in honor of the Year of Darwin, Erik Stokstad explores the question of the relative importance of the various forces that act on a biological community.
Development of Antibodies against Anthrose Tetrasaccharide for Specific Detection of
Bacillus anthracis
Spores
Authors: Kuehn Andrea , Kovác Pavol , Saksena Rina , Bannert Norbert , Klee Silke R. , Ranisch Heidrun , Grunow Roland
Published: October 2009

Methods for the immunological detection of
Bacillus anthracis
in various environmental samples and the discrimination of
B. anthracis
from other members of the
B. cereus
group are not yet well established. To generate specific discriminating antibodies, we immunized rabbits, mice, and chickens with inactivated
B. anthracis
spores and, additionally, immunized rabbits and mice with the tetrasaccharide β-Ant-(1→3)-α-
l
-Rhap-(1→3)-α-
l
-Rhap-(1→2)-
l
-Rhap. It is a constituent of the exosporium glycoprotein BclA and contains the newly discovered sugar anthrose 2-
O
-methyl-4-(3-hydroxy-3-methylbutamido)-4,6-dideoxy-β-
d
-glucose. The BclA protein is a major component of the exosporium of
B. anthracis
spores and is decorated by the tetrasaccharide indicated above. The anthrose-containing tetrasaccharide chain seems to be highly specific for
B. anthracis
, which makes it a key biomarker for the detection of these spores. The different immunizations led to anthrose-reactive polyclonal and monoclonal antibodies which were analyzed by various methods to characterize their ability to discriminate between
B. anthracis
and other
Bacillus
spp. Multiple applications, such as enzyme-linked immunosorbent assay, indirect immunofluorescence assay, and electron microscopy, revealed the specificities of the polyclonal and monoclonal antibodies generated for
B. anthracis
spore detection. All polyclonal antibodies were able to correctly identify the
B. anthracis
strains tested and showed only minimal cross-reactivities with other
Bacillus
strains. Moreover, the antibodies generated proved functional in a new capture assay for
B. anthracis
spores and could therefore be useful for the detection of spores in complex samples.
Comparison of Five Commercial Anti-Tetanus Toxoid Immunoglobulin G Enzyme-Linked Immunosorbent Assays
Authors: Perry A. L. , Hayes A. J. , Cox H. A. , Alcock F. , Parker A. R.
Published: October 2009

Five commercially available enzyme-linked immunosorbent assays for the measurement of anti-tetanus toxoid immunoglobulin G (IgG) antibodies were evaluated for performance. The data suggest that there are manufacturer-dependent differences in sensitivity and accuracy for the determination of tetanus toxoid IgG antibodies that could result in different diagnostic interpretations.
Activity of MGCD290, a Hos2 Histone Deacetylase Inhibitor, in Combination with Azole Antifungals against Opportunistic Fungal Pathogens
Authors: Pfaller M. A. , Messer S. A. , Georgopapadakou N. , Martell L. A. , Besterman J. M. , Diekema D. J.
Published: October 2009

We report on the in vitro activity of the Hos2 fungal histone deacetylase (HDAC) inhibitor MGCD290 (MethylGene, Inc.) in combination with azoles against azole-resistant yeasts and molds. Susceptibility testing was performed by the CLSI M27-A3 and M38-A2 broth microdilution methods. Testing of the combinations (MGCD290 in combination with fluconazole, posaconazole, or voriconazole) was performed by the checkerboard method. The fractional inhibitory concentrations were determined and were defined as <0.5 for synergy, ≥0.5 but <4 for indifference, and ≥4 for antagonism. Ninety-one isolates were tested, as follows: 30
Candida
isolates, 10
Aspergillus
isolates, 15 isolates of the Zygomycetes order, 10
Cryptococcus neoformans
isolates, 8
Rhodotorula
isolates, 8
Fusarium
isolates, 5
Trichosporon
isolates, and 5
Scedosporium
isolates. MGCD290 showed modest activity when it was used alone (MICs, 1 to 8 μg/ml) and was mostly active against azole-resistant yeasts, but the MICs against molds were high (16 to >32 μg/ml). MGCD290 was synergistic with fluconazole against 55 (60%) of the 91 isolates, with posaconazole against 46 (51%) of the 91 isolates, and with voriconazole against 48 (53%) of the 91 isolates. Synergy between fluconazole and MGCD290 was observed against 26/30 (87%)
Candida
isolates. All 23 of the 91
Candida
isolates that were not fluconazole susceptible demonstrated a reduced fluconazole MIC that crossed an interpretive breakpoint (e.g., resistant [MIC, ≥64 μg/ml] to susceptible [MIC, ≤8 μg/ml]) when fluconazole was combined with MGCD290 at 0.12 to 4 μg/ml. The activity of fluconazole plus MGCD290 was also synergistic against 6/10
Aspergillus
isolates. Posaconazole plus MGCD290 demonstrated synergy against 14/15 Zygomycetes (9
Rhizopus
isolates and 5
Mucor
isolates). Voriconazole plus MGCD290 demonstrated synergy against six of eight
Fusarium
isolates. Thus, MGCD290 demonstrated in vitro synergy with azoles against the majority of clinical isolates tested, including many azole-resistant isolates and genera inherently resistant to azoles (e.g.,
Mucor
and
Fusarium
). Further evaluation of fungal HDAC inhibitor-azole combinations is indicated.
Typing of
Campylobacter jejuni
Isolates from Dogs by Use of Multilocus Sequence Typing and Pulsed-Field Gel Electrophoresis
Authors: Parsons B. N. , Cody A. J. , Porter C. J. , Stavisky J. H. , Smith J. L. , Williams N. J. , Leatherbarrow A. J. H. , Hart C. A. , Gaskell R. M. , Dingle K. E. , Dawson S.
Published: October 2009

Campylobacter
is a major cause of human gastroenteritis worldwide. Risk of
Campylobacter
infection in humans has been associated with many sources, including dogs. This study aimed to investigate whether
C. jejuni
carried by dogs could potentially be a zoonotic risk for humans and if there were common sources of
C. jejuni
infection for both humans and dogs. Multilocus sequence typing (MLST) together with macrorestriction analysis of genomic DNA using SmaI and pulsed-field gel electrophoresis (PFGE) were both used to analyze 33
C. jejuni
isolates obtained from various dog populations, including those visiting veterinary practices and from different types of kennels. MLST data suggested that there was a large amount of genetic diversity between dog isolates and that the majority of sequence types found in isolates from these dogs were the same as those found in isolates from humans. The main exception was ST-2772, which was isolated from four samples and could not be assigned to a clonal complex. The most commonly identified clonal complex was ST-45 (11 isolates), followed by ST-21 (4 isolates), ST-508 (4 isolates), and ST-403 (3 isolates). The profiles obtained by macrorestriction PFGE were largely in concordance with the MLST results, with a similar amount of genetic diversity found. The diversity of sequence types found within dogs suggests they are exposed to various sources of
C. jejuni
infection. The similarity of these sequence types to
C. jejuni
isolates from humans suggests there may be common sources of infection for both dogs and humans. Although only a small number of household dogs may carry
C. jejuni
, infected dogs should still be considered a potential zoonotic risk to humans, particularly if the dogs originate from kennelled or hunt kennel dog populations, where the prevalence may be higher.
Frequent Detection of Highly Diverse Variants of
Cardiovirus
,
Cosavirus
,
Bocavirus
, and
Circovirus
in Sewage Samples Collected in the United States
Authors: Blinkova O. , Rosario K. , Li L. , Kapoor A. , Slikas B. , Bernardin F. , Breitbart M. , Delwart E.
Published: October 2009

Untreated sewage samples from 12 cities in the United States were screened for the presence of recently characterized RNA and DNA viruses found at high prevalence in the stool specimens of South Asian children. Genetic variants of human cosaviruses and cardioviruses in the
Picornaviridae
family and of DNA circoviruses and human bocaviruses were detected, expanding the known genetic diversity and geographic range of these newly identified viruses. All four virus groups were detected in sewage samples of less than a milliliter from multiple U.S. cities. PCR screening of particle-protected viral nucleic acid in sewage samples could therefore rapidly establish the presence and determine the diversity of four newly described enteric viruses in large urban populations. More frequent and deeper sampling of viral nucleic acids in sewage samples could be used to monitor changes in the prevalence and genetic composition of these and other novel enteric viruses.
Identification ofCryptococcus gattiiby Use ofl-Canavanine Glycine Bromothymol Blue Medium and DNA Sequencing
Authors: Klein K. R. , Hall L. , Deml S. M. , Rysavy J. M. , Wohlfiel S. L. , Wengenack N. L.
Published: October 2009

Cryptococcus neoformansandCryptococcus gattiiare closely related pathogenic fungi.Cryptococcus neoformansis ecologically widespread and affects primarily immunocompromised patients, whileC. gattiiis traditionally found in tropical climates and has been reported to cause disease in immunocompetent patients.l-Canavanine glycine bromothymol blue (CGB) agar can be used to differentiateC. neoformansandC. gattii, but there are few reports of its performance in routine clinical practice. Growth ofC. gattiion CGB agar produces a blue color, indicating the assimilation of glycine, whileC. neoformansfails to cause a color change. Using reference and clinical strains, we evaluated the ability of CGB agar and D2 large ribosomal subunit DNA sequencing (D2 LSU) to differentiateC. neoformansandC. gattii.One hundred two yeast isolates were screened for urease activity, melanin production, and glycine assimilation on CGB agar as well as by D2 sequencing. Seventeen of 17 (100%)C. gattiiisolates were CGB positive, and 54 of 54C. neoformansisolates were CGB negative. Several yeast isolates other than theC. gattiiisolates were CGB agar positive, indicating that CGB agar cannot be used alone for identification ofC. gattii.D2 correctly identified and differentiated allC. gattiiandC. neoformansisolates. This study demonstrates that the use of CGB agar, in conjunction with urea hydrolysis and Niger seed agar, or D2 LSU sequencing can be reliably used in the clinical laboratory to distinguishC. gattiifromC. neoformans. We describe how CGB agar and D2 sequencing have been incorporated into the yeast identification algorithm in our laboratory.
Comparison of a Rapid Molecular Method, the BD GeneOhm Cdiff Assay, to the Most Frequently Used Laboratory Tests for Detection of Toxin-Producing
Clostridium difficile
in Diarrheal Feces
Authors: Terhes Gabriella , Urbán Edit , Sóki József , Nacsa Enikő , Nagy Elisabeth
Published: October 2009

Six hundred diarrheal stool specimens were collected from inpatients and outpatients at local university hospitals for the detection of toxigenic
Clostridium difficile
using three parallel methods, the BD GeneOhm Cdiff assay, the tissue culture cytotoxicity assay, and a commercially available enzyme-linked fluorescence immunoassay (ELFA) (Vidas
C. difficile
toxin A and B assay; bioMérieux). Toxigenic
C. difficile
culture was also performed to further clarify discordant results. During a 3-month study period, 58 (9.7%) of the 600 diarrheal samples examined were positive by the BD GeneOhm Cdiff assay, while the Vidas
C. difficile
toxin A and B assay and the cytotoxicity assay performed directly on stool samples gave 4.7% and 6.3% positivity rates, respectively. In the case of four samples, BD GeneOhm Cdiff assay results were not evaluable at first because of the presence of PCR inhibitors, but upon repeat testing from the frozen lysates, all of these samples proved to be negative. After resolution with toxigenic culture, the cytotoxicity assay proved to be positive in 55 samples (9.2%), while the ELFA was positive in 37 samples (6.2%). Results of culture and repeated cytotoxicity assays emphasized the importance of the culture method, because the use of ELFA or enzyme immunoassay without a culture method may lead to a substantial portion of toxigenic
C. difficile
strains being missed.
Diagnosis of Endotoxemia with Gram-Negative Bacteremia Is Bacterial Species Dependent: a Meta-Analysis of Clinical Studies
Authors: Hurley James C.
Published: October 2009

Endotoxemia is undetectable for up to 60% of cases of bacteremia caused by gram-negative (GN) species, a discordance attributed to the limitations of theLimulusassay for endotoxemia. The lipid A structure of the endotoxin molecule is critical for the sensing of GN bacteria by the host immune system although not so for sensing by theLimulusassay. The lipid A structure of commensalEnterobacteriaceaeis hexa-acyl, whereas non-Enterobacteriaceaehave a broader range of structures. By using a previously published classification of lipid A structures (R. S. Munford, Infect. Immun. 76:454-465, 2008), the association of endotoxemia with bacteremia caused by GN organisms is reexamined for 580 GN bacteremic patients from 46 studies. Endotoxemia was less commonly detected for cases of bacteremia caused bySalmonellaentericaserovar Typhi (four studies; 15 of 55 cases of bacteremia [27%]) than for cases of bacteremia caused byNeisseria meningitidis(five studies; 69 of 84 cases [82%]) andPseudomonas pseudomallei(one study; 38 of 41 cases [93%]) among studies restricted to those with specified cases of bacteremia caused by GN organisms. Among 23 unrestricted studies, endotoxemia was less commonly detected for cases of bacteremia with a commensal member of theEnterobacteriaceae(104 of 240 cases [43%]) than with non-Enterobacteriaceae(59 of 100 cases [59%]) (summary odds ratio, 0.53 [90% confidence interval, 0.33 to 0.85]). This finding is consistent across all the unrestricted studies, even including studies with seemingly contrary results for endotoxemia diagnosis among cases of bacteremia caused by GN bacteria overall. Surprisingly, with bacteremia caused by commensalEnterobacteriaceae, the diagnosis of endotoxemia appears to be unrelated to theLimulusassay sensitivity. Across these 45 studies, the association of endotoxemia with GN bacteremia is variable but consistent for different types of GN bacteremia.
Failure of the BD GeneOhm StaphSR Assay for Direct Detection of Methicillin-Resistant and Methicillin-Susceptible
Staphylococcus aureus
Isolates in Positive Blood Cultures Collected in the United States
Authors: Snyder James W. , Munier Gina K. , Heckman Stacy A. , Camp Pamela , Overman Timothy L.
Published: October 2009

Fifty-nine
Staphylococcus aureus
isolates from one hundred blood cultures containing gram-positive cocci in clusters were identified by conventional methods and the BD GeneOhm StaphSR assay (SR). The SR misidentified three methicillin (meticillin)-resistant
S. aureus
(MRSA) isolates as methicillin-susceptible
S. aureus
(MSSA), while one MSSA isolate tested negative for
S. aureus
. The three MRSA isolates were strains with MREJ types that cannot be detected by the currently available SR.
Screening of a Large Global
Aspergillus fumigatus
Species Complex Collection by Using a Species-Specific Microsphere-Based Luminex Assay
Authors: Etienne Kizee A. , Gade Lalitha , Lockhart Shawn R. , Diekema Daniel J. , Messer Shawn A. , Pfaller Michael A. , Balajee S. Arunmozhi
Published: October 2009

A microsphere-based Luminex assay was developed and validated for rapid identification of
Aspergillus fumigatus
from the other species within the
A. fumigatus
species complex (section
Fumigati
). This molecular tool was then employed to screen 499 clinical
A. fumigatus
species complex isolates collected from multiple medical centers throughout the world with results demonstrating the exclusive presence of
A. fumigatus
.
Detection of Bocavirus in Saliva of Children with and without Respiratory Illness
Authors: Martin Emily T. , Taylor James , Kuypers Jane , Magaret Amalia , Wald Anna , Zerr Danielle , Englund Janet A.
Published: October 2009

We evaluated saliva samples from 149 children 2 to 11 years old for human bocavirus (hBoV) DNA. hBoV was detected in saliva samples at asymptomatic enrollment in 3% (5/149) and during respiratory illness in 2% (2/106) of the cases. hBoV was detected in only 1/149 asymptomatic and 0/106 illness nasal samples.
Identification and Differentiation of Clinically Relevant Mycobacterium Species Directly from Acid-Fast Bacillus-Positive Culture Broth
Authors: Li Haijing , Turhan Vedat , Chokhani Laxmi , Stratton Charles W. , Dunbar Sherry A. , Tang Yi-Wei
Published: October 2009

Mycobacterium
species cause a variety of clinical diseases, some of which may be species specific. Therefore, it is clinically desirable to rapidly identify and differentiate mycobacterial isolates to the species level. We developed a rapid and high-throughput system, MycoID, to identify
Mycobacterium
species directly from acid-fast bacillus (AFB)-positive mycobacterial culture broth. The MycoID system incorporated broad-range PCR followed by suspension array hybridization to identify 17 clinically relevant mycobacterial complexes, groups, and species in one single reaction. We evaluated a total of 271 AFB-positive culture broth specimens, which were identified by reference standard methods in combination with biochemical and molecular tests. The overall identification agreement between the standard and the MycoID system was 89.7% (perfect match) or 97.8% (one match in codetection). In comparison to the standard, the MycoID system possessed an overall sensitivity of 97.1% and specificity of 98.8%. The 159
Mycobacterium avium-M. intracellulare
complex isolates were further identified to the species level by MycoID as being
M. avium
(
n
= 98; 61.1%),
M. intracellulare
(
n
= 57; 35.8%), and mixed
M. avium
and
M. intracellulare
(
n
= 2; 1.3%).
M. avium
was recovered more frequently from sterile sites than
M. intracellulare
(odds ratio, 4.6;
P
= 0.0092). The entire MycoID procedure, including specimen processing, can be completed within 5 h, providing rapid and reliable identification and differentiation of mycobacterium species that is amenable to automation. Additional differentiation of
Mycobacterium avium-M. intracellulare
complex strains into
M. avium
and
M. intracellulare
may provide a tool to better understand the role of
Mycobacterium avium-M. intracellulare
complex isolates in human disease.
Acute Infection with Venezuelan Equine Encephalitis Virus Replicon Particles Catalyzes a Systemic Antiviral State and Protects from Lethal Virus Challenge
Authors: Konopka Jennifer L. , Thompson Joseph M. , Whitmore Alan C. , Webb Drue L. , Johnston Robert E.
Published: October 2009

The host innate immune response provides a critical first line of defense against invading pathogens, inducing an antiviral state to impede the spread of infection. While numerous studies have documented antiviral responses within actively infected tissues, few have described the earliest innate response induced systemically by infection. Here, utilizing Venezuelan equine encephalitis virus (VEE) replicon particles (VRP) to limit infection to the initially infected cells in vivo, a rapid activation of the antiviral response was demonstrated not only within the murine draining lymph node, where replication was confined, but also within distal tissues. In the liver and brain, expression of interferon-stimulated genes was detected by 1 to 3 h following VRP footpad inoculation, reaching peak expression of >100-fold over that in mock-infected animals. Moreover, mice receiving a VRP footpad inoculation 6, 12, or 24 h prior to an otherwise lethal VEE footpad challenge were completely protected from death, including a drastic reduction in challenge virus titers. VRP pretreatment also provided protection from intranasal VEE challenge and extended the average survival time following intracranial challenge. Signaling through the interferon receptor was necessary for antiviral gene induction and protection from VEE challenge. However, VRP pretreatment failed to protect mice from a heterologous, lethal challenge with vesicular stomatitis virus, yet conferred protection following challenge with influenza virus. Collectively, these results document a rapid modulation of the host innate response within hours of infection, capable of rapidly alerting the entire animal to pathogen invasion and leading to protection from viral disease.
Populations of Genomic RNAs Devoted to the Replication or Spread of a Bipartite Plant Virus Differ in Genetic Structure
Authors: Lozano Gloria , Grande-Pérez Ana , Navas-Castillo Jesús
Published: October 2009

RNA viruses within a host exist as dynamic distributions of closely related mutants and recombinant genomes. These closely related mutants and recombinant genomes, which are subjected to a continuous process of genetic variation, competition, and selection, act as a unit of selection, termed viral quasispecies. Characterization of mutant spectra within hosts is essential for understanding viral evolution and pathogenesis resulting from the cooperative behavior of viral mutants within viral quasispecies. Furthermore, a detailed analysis of viral variability within hosts is needed to design control strategies, because viral quasispecies are reservoirs of viral variants that potentially can emerge with increased virulence or altered tropism. In this work, we report a detailed analysis of within-host viral populations in 13 field isolates of the bipartiteTomato chlorosis virus(ToCV) (genusCrinivirus, familyClosteroviridae). The intraisolate genetic structure was analyzed based on sequencing data for 755 molecular clones distributed in four genomic regions within the RNA-dependent RNA polymerase (RNA1) and Hsp70h, CP, and CPm (RNA2) open reading frames. Our results showed that populations of ToCV within a host plant have a heterogeneous and complex genetic structure similar to that described for animal and plant RNA viral quasispecies. Moreover, the structures of these populations clearly differ depending on the RNA segment considered, being more complex for RNA1 (encoding replication-associated proteins) than for RNA2 (encoding encapsidation-, systemic-movement-, and insect transmission-relevant proteins). These results support the idea that, in multicomponent RNA viruses, function can generate profound differences in the genetic structures of the different genomic segments.
The 5′ Leader of the mRNA Encoding the Marek's Disease Virus Serotype 1 pp14 Protein Contains an Intronic Internal Ribosome Entry Site with Allosteric Properties
Authors: Tahiri-Alaoui Abdessamad , Matsuda Daiki , Xu Hongtao , Panagiotis Panopoulos , Burman Luke , Lambeth Luke S. , Petherbridge Lawrence , James William , Mauro Vincent , Nair Venugopal
Published: October 2009

We demonstrate the presence of a functional internal ribosome entry site (IRES) within the 5′ leader (designated 5L) from a variant of bicistronic mRNAs that encode the pp14 and RLORF9 proteins from Marek's disease virus (MDV) serotype 1. Transcribed as a 1.8-kb family of immediate-early genes, the mature bicistronic mRNAs have variable 5′ leader sequences due to alternative splicing or promoter usage. Consequently, the presence or absence of the 5L IRES in the mRNA dictates the mode of pp14 translation and leads to the production of two pp14 isoforms that differ in their N-terminal sequences. Real-time reverse transcription-quantitative PCR indicates that the mRNA variants with the 5L IRES is two to three times more abundant in MDV-infected and transformed cells than the mRNA variants lacking the 5L IRES. A common feature to all members of the 1.8-kb family of transcripts is the presence of an intercistronic IRES that we have previously shown to control the translation of the second open reading frame (i.e., RLORF9). Investigation of the two IRESs residing in the same bicistronic reporter mRNA revealed functional synergism for translation efficiency. In analogy with allosteric models in proteins, we propose IRES allostery to describe such a novel phenomenon. The functional implications of our findings are discussed in relation to host-virus interactions and translational control.
NKG2D Ligand MICA Is Retained in thecis-Golgi Apparatus by Human Cytomegalovirus Protein UL142
Authors: Ashiru Omodele , Bennett Neil J. , Boyle Louise H. , Thomas Mair , Trowsdale John , Wills Mark R.
Published: October 2009

Human cytomegalovirus (HCMV) evades T-cell recognition by down-regulating expression of major histocompatibility complex (MHC) class I and II molecules on the surfaces of infected cells. Contrary to the “missing-self” hypothesis, HCMV-infected cells are refractory to lysis by natural killer (NK) cells. Inhibition of NK cell function is mediated by a number of HCMV immune evasion molecules, which operate by delivering inhibitory signals to NK cells and preventing engagement of activating ligands. One such molecule is UL142, which is an MHC class I-related glycoprotein encoded by clinical isolates and low-passage-number strains of HCMV. UL142 is known to down-modulate surface expression of MHC class I-related chain A (MICA), which is a ligand of the activating NK receptor NKG2D. However, the mechanism by which UL142 interferes with MICA is unknown. Here, we show that UL142 localizes predominantly to the endoplasmic reticulum (ER) andcis-Golgi apparatus. The transmembrane domain of UL142 mediates its ER localization, while we propose that the UL142 luminal domain is involved in itscis-Golgi localization. We also confirm that UL142 down-modulates surface expression of full-length MICA alleles while having no effect on the truncated allele MICA*008. However, we demonstrate for the first time that UL142 retains full-length MICA alleles in thecis-Golgi apparatus. In addition, we propose that UL142 interacts with nascent MICA en route to the cell surface but not mature MICA at the cell surface. Our data also demonstrate that the UL142 luminal and transmembrane domains are involved in recognition and intracellular sequestration of full-length MICA alleles.
Insertion of Enhanced Green Fluorescent Protein in a Hinge Region of Vesicular Stomatitis Virus L Polymerase Protein Creates a Temperature-Sensitive Virus That Displays No Virion-Associated Polymerase Activity In Vitro
Authors: Ruedas John B. , Perrault Jacques
Published: October 2009

The RNA-dependent RNA polymerase of viruses belonging to the order
Mononegavirales
is part of a large multifunctional L protein that also catalyzes viral mRNA capping and cap methylation. The L protein of this diverse group of agents displays six blocks of conserved sequences. The precise relationship between these conserved regions and individual functions is largely unknown, except for “domain” VI that clearly encodes a viral mRNA cap methylase. The L protein of morbilliviruses (family
Paramyxoviridae
) was reported to tolerate insertion of the enhanced green fluorescent protein (EGFP) in a region just upstream of domain VI. Recombinant viruses with this insertion grow well in cell culture but are highly attenuated in animal hosts. We show here that the L protein of vesicular stomatitis virus (VSV), the prototype of the
Rhabdoviridae
family, also tolerates insertion of EGFP at a similar site. The modified protein (L
EGFP
) and the resultant recombinant virus both demonstrated a sharp temperature-sensitive phenotype for polymerase activity, with reduced activity at 37°C and no activity at 37.5°C. Neither translation nor methylation of mutant virus transcripts was affected at 37°C. Curiously, mutant virus grown at permissive temperature contained about threefold-less L protein than the wild-type virus did and displayed no virion-associated polymerase activity in vitro. These findings support the notion that a flexible “hinge” region separates the cap methylase domain of L proteins from upstream functions and open up a number of avenues for studies of L-protein function in the more-tractable VSV model system.
The Amino Terminus of Herpes Simplex Virus Type 1 Glycoprotein K (gK) Modulates gB-Mediated Virus-Induced Cell Fusion and Virion Egress
Authors: Chouljenko Vladimir N. , Iyer Arun V. , Chowdhury Sona , Chouljenko Dmitry V. , Kousoulas Konstantin G.
Published: October 2009

Herpes simplex virus type 1 (HSV-1)-induced cell fusion is mediated by viral glycoproteins and other membrane proteins expressed on infected cell surfaces. Certain mutations in the carboxyl terminus of HSV-1 glycoprotein B (gB) and in the amino terminus of gK cause extensive virus-induced cell fusion. Although gB is known to be a fusogenic glycoprotein, the mechanism by which gK is involved in virus-induced cell fusion remains elusive. To delineate the amino-terminal domains of gK involved in virus-induced cell fusion, the recombinant viruses gKΔ31-47, gKΔ31-68, and gKΔ31-117, expressing gK carrying in-frame deletions spanning the amino terminus of gK immediately after the gK signal sequence (amino acids [aa] 1 to 30), were constructed. Mutant viruses gKΔ31-47 and gKΔ31-117 exhibited a gK-null (ΔgK) phenotype characterized by the formation of very small viral plaques and up to a 2-log reduction in the production of infectious virus in comparison to that for the parental HSV-1(F) wild-type virus. The gKΔ31-68 mutant virus formed substantially larger plaques and produced 1-log-higher titers than the gKΔ31-47 and gKΔ31-117 mutant virions at low multiplicities of infection. Deletion of 28 aa from the carboxyl terminus of gB (gBΔ28syn) caused extensive virus-induced cell fusion. However, the gBΔ28syn mutation was unable to cause virus-induced cell fusion in the presence of the gKΔ31-68 mutation. Transient expression of a peptide composed of the amino-terminal 82 aa of gK (gKa) produced a glycosylated peptide that was efficiently expressed on cell surfaces only after infection with the HSV-1(F), gKΔ31-68, ΔgK, or UL20-null virus. The gKa peptide complemented the gKΔ31-47 and gKΔ31-68 mutant viruses for infectious-virus production and for gKΔ31-68/gBΔ28syn-mediated cell fusion. These data show that the amino terminus of gK modulates gB-mediated virus-induced cell fusion and virion egress.