Comparison of different RT-qPCR assays for the detection of human and bovine group A rotaviruses and characterization by sequences analysis of genes encoding VP4 and VP7 capsid proteins.

AIMS: The aim of this study was to compare the performance of four RT-qPCR assays for the detection of human and bovine group A rotaviruses and to characterize the positive samples by sequence analysis of VP4 and VP7 genes. METHODS AND RESULTS: RNA extracted from eight human rotavirus strains, and a panel of 33 human and 25 bovine faecal samples was subjected to different RT-qPCR detection systems. Among these assays, only RT-qPCR primers and probe systems B and C were able to detect all human rotavirus strains from cell culture solutions and faecal samples. However, the results showed that the system C was generally more sensitive by one or two logs than the other RT-qPCR assays tested. With the bovine faecal samples, the most efficient RT-qPCR systems were B and A with the detection in 100 and 92% of samples tested, respectively. Human group A rotavirus G1P[8] and bovine G6P[11] were the most frequently used strains identified in this study. A G3P[9] strain, closely related to a feline rotavirus isolated in the USA, was also discovered in a human rotavirus infection. CONCLUSION: The RT-qPCR system B was the only TaqMan assay evaluated in this study able to detect rotavirus RNA in all positive human and bovine faecal samples. SIGNIFICANCE AND IMPACT OF THE STUDY: Utilization of only one RT-qPCR for the detection of human and bovine group A rotaviruses and the possibility of human infection by a feline rotavirus strain.

Development of a real-time TaqMan PCR assay for the detection of porcine and bovine Torque teno virus.

AIMS: The goal of this study was to develop and to optimize molecular tools to detect the presence of Torque teno virus (TTV) in swine and cattle. A novel real-time polymerase chain reaction (PCR) using a TaqMan probe was developed to detect both genogroups of TTV strains. METHODS AND RESULTS: Oligonucleotide primers and hybridization probes were designed based on sequence analysis of the noncoding region, a highly conserved part of the genome. The real-time PCR assay specifically detected bovine and porcine TTV DNA without cross-amplification of other common pathogens. The assay was compared with conventional PCR and nested-PCR assays for the detection of porcine genogroups 1 and 2 and bovine TTV on plasma and faecal samples, and the assay was found faster, more reliable and reduced the risk of false positive results. CONCLUSIONS: The real-time PCR assay provided better detection results for the two TTV genogroups in both swine and cattle compared to the conventional PCR assays. SIGNIFICANCE AND IMPACT OF THE STUDY: This new TaqMan PCR assay will be a useful tool for the detection of animal TTV strains, to evaluate the viral load from animal host and finally to identify the presence of these viruses in the agri-food continuum.

Comparative analysis of different TaqMan real-time RT-PCR assays for the detection of swine Hepatitis E virus and integration of Feline calicivirus as internal control.

AIMS: The aim of this study was to compare the performance of four TaqMan RT-PCR assays with a commonly used nested RT-PCR and to include the Feline calicivirus (FCV) as an internal control. METHODS AND RESULTS: RNA extracted from 87 swine faecal samples and 103 swine blood samples was subjected to different detection systems. Faecal samples naturally contaminated with Hepatitis E virus (HEV) and negative samples were artificially inoculated with 3.2 x 10(3) PFU of FCV. Detection results obtained on faecal and plasma samples were 35.6% and 4.9% with the nested RT-PCR assay, 8.0% and 0%, 0% and 0%, 13.8% and 0% and 36.8% and 3.9% with TaqMan systems A, B, C and D respectively. The Ct means obtained with the multiplex TaqMan assay were 30.11 and 30.43 for the detection of FCV with HEV contaminated samples and negative samples. CONCLUSIONS: The TaqMan system D was more suitable for the detection of swine HEV strains than the three others and FCV was integrated successfully as an internal control. SIGNIFICANCE AND IMPACT OF THE STUDY: FCV was demonstrated as an efficient control to monitor the RNA extraction process and HEV amplification procedure in a multiplex HEV/FCV TaqMan assay. This control would be helpful in limiting false negative results.

Molecular detection of bovine and porcine Torque teno virus in plasma and feces.

Torque teno virus (TTV) is frequently detected in humans, livestock and some companion animals. Very little is known about presence of TTV in Canadian livestock and the goal of this study was to evaluate the presence of TTV in swine and cattle using molecular tools. TTV DNA was detected and confirmed by sequencing in the plasma of 90.5% and in the feces of 60.3% of the animals tested in a single swine herd as well as 80.9% and 1.1% in the plasma of individuals from general Quebec swine and cattle populations, respectively. The impact of the TTV presence in livestock population for the agri-food chain should be further investigated.

Nano-micro structured superhydrophobic zinc coating on steel for prevention of corrosion and ice adhesion.

Thin films of zinc have been deposited on steel substrates by electrodeposition process and further functionalized with ultra-thin films of commercial silicone rubber, in order to obtain superhydrophobic properties. Morphological feature, by scanning electron microscope (SEM), shows that the electrodeposited zinc films are composed of micro-nano rough patterns. Furthermore, chemical compositions of these films have been analyzed by X-ray diffraction (XRD) and infra-red (IRRAS). An optimum electrodeposition condition, based on electrical potential and deposition time, has been obtained which provides superhydrophobic properties with a water contact angle of 155±1°. The corrosion resistance properties, in artificial seawater, of the superhydrophobic zinc coated steel are found to be superior to bare steel. Similarly, the measured ice adhesion strength on superhydrophobic surfaces, using the centrifugal adhesion test (CAT), is found to be 6.3 times lower as compared to bare steel. This coating has promising applications in offshore environment, to mitigate corrosion and reduce ice adhesion.

Evaluation of the buccal bleeding time and platelet glass bead retention as assays of hemostasis in the dog: the effects of acetylsalicylic acid, warfarin and von Willebrand factor deficiency.

The study evaluated two hemostatic assays in the dog, a modified version of the buccal mucosal bleeding time (BMBT) and the platelet glass bead retention (PR), to describe the aspects of hemostasis measured by these assays. Von Willebrand factor (vWf)-deficient Doberman pinscher dogs were used in evaluating the effects of altered platelet adhesion. Normal dogs were treated with either acetylsalicylic acid (ASA) or warfarin to evaluate the effects of altered platelet aggregation and coagulation. There was significant prolongation of the BMBT and reduction of the PR in vWf-deficient dogs as compared to normal dogs. In ASA treated dogs the BMBT was slightly prolonged; the PR was significantly reduced. The change in ASA-induced BMBT did not correlate with the sensitivity of the dog platelets to arachidonic acid. In warfarin treated dogs there was no change in the BMBT; however, the PR was significantly reduced. The BMBT is a test of hemostasis that is sensitive to platelet adhesion and aggregation deficits. The PR is useful in detecting general abnormalities in hemostasis including platelet adhesion defects due to reduced vWf.

Unilateral absence of a pulmonary artery. Data from cardiopulmonary exercise testing.

A case of congenital, unilateral absence of a left pulmonary artery is described in a patient presenting with exertional dyspnea. Data from cardiopulmonary exercise testing suggest that the mechanism of dyspnea is secondary to a paradoxic elevation of the physiologic dead space to tidal volume ratio (VD/VT) during exercise.

Varicella vaccination for healthcare workers at a university hospital: an analysis of costs and benefits.

OBJECTIVE: To demonstrate the costs and benefits of vaccinating varicella-susceptible healthcare workers at a university hospital with live, attenuated varicella-zoster virus vaccine. DESIGN: Retrospective review of employee medical records and data on the cost of special paid absence for susceptible healthcare workers after exposure to varicella or herpes zoster. SETTING: A 988-bed tertiary-care university hospital. RESULTS: In 1994, 224 hospital employees (3.4%) were susceptible to the varicella-zoster virus. There were 40 exposures to varicella and herpes zoster in that year, involving 29 of the susceptible employees. Nine (31%) of the exposed susceptibles became varicella immune by indirect fluorescent antibody testing subsequent to exposure. Seventeen (59%) have had multiple varicella exposures and special paid absences while employed by the hospital. In 1994, wages paid to healthcare workers while furloughed for the communicable period following varicella exposure totaled $38,463.93. An additional $24,748.74 was paid to replacement workers during that same time. Varicella vaccine to immunize all 224 susceptibles in 1994 would have cost $17,920. Absences due to varicella and herpes zoster exposure also result in disruptions to patient care. CONCLUSIONS: Varicella vaccination for varicella-susceptible healthcare workers at a university hospital would result in financial savings and improved patient care. We recommend that other institutions consider the costs and benefits of adopting a varicella immunization program for their susceptible employees.

A selective 5-HT(1) agonist, RU 24969, increases locus coeruleus catechol metabolic and neuronal activity.

Locus coeruleus activity was monitored by either in vivo electrochemistry, post-mortem HPLC, or single unit activity, after systemic administration of RU 24969, a potent serotonin-1 agonist. Whatever the methodology, activation of the locus coeruleus appeared after RU 24969 injection. Catechol oxidation current, assessed by in vivo differential pulse voltammetry and single unit activity in the locus coeruleus showed simultaneous increases after RU 24969. The increase in catechol oxidation current after RU 24969 was dose dependent (ED(50) = 1.4 mg kg(?1) of i.p. RU 24969). This increased activity was also observed on microdissected locus coeruleus as shown by the increased levels of dopamine and 3,4-dihydroxyphenylacetic acid measured with high performance liquid chromatography. Furthermore, RU 24969 treatment decreased 5-hydroxyindolacetic acid/serotonin ratio in the same microdissected locus coeruleus. This increased locus coeruleus catechol metabolic activity was suppressed by making lesions in the serotonergic systems with 5,7-dihydroxytryptamine. By contrast, neither 8-OHDPAT nor methysergide produced significant changes in the catechol oxidation current recorded in the locus coeruleus.

Effects of hyperoxia on rat diaphragm function.

The association of oxygen radical generation with impaired diaphragm performance has previously been reported after inspiratory resistive loading (IRL). We hypothesized that exposure of rats to normobaric hyperoxia (O2) could produce impaired diaphragm function because of free radical production. Sprague-Dawley rats were divided into four groups: 1) room air (control), 2) > 95% O2 for 24 h, 3) > 95% O2 for 48 h, and 4) > 95% O2 for 60 h. Each group was studied at rest after the O2 exposure and then after IRL. During IRL, the animals breathed through an inspiratory resistor until they were unable to sustain > 70% of the maximum airway pressure. Diaphragm samples were obtained for analysis of glutathione (GSH) and glutathione disulfide (GSSG) concentrations. In vitro isometric contractile properties were also determined, including maximal tetanic tension (Po) and maximal twitch tension (Pt), in GSSG content and in GSSG-to-GSH ratios. Hyperoxia for > 48 h resulted in significant decreases in Po and Pt and an increase in GSSG content and in GSSG-to-GSH ratios compared with other groups. Those same animals subjected to IRL showed a further decrease in Po and Pt. These data suggest that free radical generation may occur in the diaphragm during a hyperoxia exposure associated with activation of the GSH redox cycle and impairment of diaphragm function.

Effect of exercise, DDAVP, and epinephrine on the factor VIII:C/von Willebrand factor complex in normal dogs and von Willebrand factor deficient Doberman pinscher dogs.

Endothelial cells in biopsied blood vessels from von Willebrand factor (vWf)-deficient Doberman pinscher dogs contain immunologically detectable vWf. These dogs and normal dogs were treated with DDAVP (0.6 microgram/kg) and epinephrine (0.5 microgram/kg/min for 30 minutes) and were exercised, using 5 different exercise protocols, (3-4 m/s for 5-40 minutes at 0-5% grade) to determine if treatments reported to increase plasma factor VIII:C/vWf complex in humans would elevate canine plasma vWf. Following the two most strenuous exercise conditions--30 and 40 minutes--plasma von Willebrand factor antigen (vWf:Ag) increased in normal dogs by 30% and 70%, respectively. Factor VIII:C was increased 47% by the most strenuous exercise conditions. The vWf-deficient dogs would not exercise beyond 30 minutes and neither vWf:Ag nor factor VIII:C activity increased. Following DDAVP, plasma vWf:Ag increased in the normal dogs by 47% and factor VIII:C activity was increased by 48%. Factor VIII:C activity increased by 30% in the vWf-deficient dogs, but there was only a slight change in vWf:Ag. Bleeding time decreased in 5 of 6 vWf-deficient dogs. In the normal dogs vWf:Ag increased by 14% after epinephrine infusion, but factor VIII:C activity did not change; neither parameter was altered in the vWf-deficient dogs. While the factor VIII:C/vWf:Ag complex was increased in the normal dog by exercise and DDAVP, the increase is not as pronounced as has been reported for humans. It is not known whether the poor response of the vWf-deficient dog is due to low levels of vWf in their endothelium or to a release defect.

Decrease of the adhesion of Streptococcus suis serotype 2 mutants to embryonic bovine tracheal cells and porcine tracheal rings.

Streptococcus suis is an important swine pathogen that may be present in the tonsils of pigs that show no signs of illness. Because adhesion to host cells may be important in the carrier state, this study was undertaken to investigate adhesion to host cells by S. suis mutant strains defective in expression of a 39-kDa protein. Mutant strains of S. suis were generated by transposon Tn916 mutagenesis and were tested for adhesion to embryonic bovine tracheal cells and porcine tracheal rings. Compared with the parent strain, there was a significant reduction in adherence of 3 mutant strains to both bovine tracheal cells and porcine tracheal rings.

Temporal variation and factors affecting measurement of canine von Willebrand factor.

OBJECTIVE: To determine whether canine plasma von Willebrand factor (vWf) varies between and within individuals over time and with different blood sample collection and processing procedures. ANIMALS: 26 adult dogs and 6 pups. PROCEDURE: Blood was obtained from the jugular or cephalic vein daily for 8 to 19 days and weekly for 9 to 23 weeks in adult dogs and periodically up to 180 days of age in pups. Temporal variation in vWf concentration and the effect of vascular occlusion, venipuncture site, lipemia, hemolysis, anticoagulant, storage time, freeze-thawing, and centrifugation speed on plasma vWf concentration, measured by ELISA, were determined. RESULTS: Plasma vWf concentration varied over time. In dogs with mean vWf concentration > or = 79 U/dl, the largest intraindividual range in vWf spanned 64 U/dl with daily and 53 U/dl with weekly sample collection. In dogs with mean vWf concentration < or = 24 U/dl, the largest individual variation was 12 U/dl with daily and weekly sample collection. In dogs with mean vWf concentration > or = 53 and < or = 74 U/dl, the largest intraindividual range spanned 35 U/ dl. Mean vWf concentration of pups from 3 to 180 days of age did not change. Sample hemolysis decreased mean vWf by 37%. Mean vWf concentration was 9% higher in cephalic than jugular vein samples (P = 0.056). Other sample collection/preparation procedures did not affect vWf concentration. CONCLUSION: There was substantial temporal variation in vWf concentration within individual dogs. CLINICAL RELEVANCE: Multiple tests may be necessary to obtain a reliable estimate of vWf concentration in dogs.

Effect of cryoprecipitate and plasma on plasma von Willebrand factor multimeters and bleeding time in Doberman Pinschers with type-I von Willebrand's disease.

We determined whether administration of cryoprecipitate or fresh-frozen plasma (FFP) would enhance glass bead platelet retention and shorten the bleeding time in von Willebrand factor (vWf)-deficient Doberman Pinschers. Plasma concentration of vWf was < 15% of the reference value in these dogs and, on the basis of multimeric analysis of vWf, these dogs had type-I von Willebrand's disease (vWd). Concentration of vWf in cryoprecipitate (prepared from FFP of clinically normal dogs) was enriched almost 20 times, and the preparation was a concentrate of the largest and most physiologically active multimers. Administration of a dose of cryoprecipitate calculated to increase plasma vWf concentration of recipient dogs to 50 U/dl increased plasma vWf concentration in recipient dogs to about 40 U/dl. Mean buccal mucosal bleeding time (BMBT) shortened from 6.7 minutes before treatment to 3.8 minutes at 2 hours after treatment. Cryoprecipitate from donor dogs treated with deamino-8-D-arginine vasopressin (1 micrograms/kg of body weight) effectively shortened mean BMBT from 6.4 minutes to 3.1 minutes. Administration of cryoprecipitate from vWf-deficient dogs prolonged, rather than shortened, the BMBT. After FFP (450 ml) infusion, plasma vWf concentration increased in recipient dogs, but the BMBT did not shorten. Glass bead platelet retention did not change after administration of cryoprecipitate or FFP.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of the healing of prescrotal urethrotomy incisions in the dog: sutured versus nonsutured.

Comparison was made of the healing of sutured prescrotal urethral incisions (12 dogs) with that of nonsutured incisions (6 dogs). Comparison was also made of the healing of 5-0 polyglactin 910-sutured urethral incisions (6 dogs) with that of 5-0 polydioxanone-sutured incisions (6 dogs). Three dogs from each treatment group were euthanatized 3 weeks and 6 weeks after the surgical procedure. Surgical sites were examined grossly, and urethral circumference measurements were taken at 3 locations (surgical site, 1 cm cranially, and 1 cm caudally). Transverse sections of the surgical sites were prepared and examined by light microscopy. Hemorrhage occurred postoperatively in dogs in which the incisions were not sutured. The surgical sites from the 6 dogs in which incisions healed by second intention had more fibrosis and less inflammation than did those that were sutured. There was little difference between incisions sutured with polyglactin 910 and those sutured with polydioxanone suture material. Postoperative urethral stricture formation did not occur in any of the dogs.

Rectal hemorrhage associated with vascular ectasia in a young dog.

Rectal bleeding in a 7-month-old 13-kg sexually intact female mixed-breed dog was determined to be associated with vascular ectasia of the small intestine, descending colon, rectum, and anus. Microscopically, the telangiectasia was associated with lymphangiectasia and focal ulceration. Surgical intervention resulted in incomplete resection of the lesion and only temporary amelioration of clinical signs. The dog's age was compatible with a congenital origin for the defect, but an acquired cause could not be excluded.

Computerizing patient questionnaires.

Hillcrest Baptist Medical Center spent two years perfecting its computerized patient questionnaire system. A look at how the system evolved illustrates some valuable lessons on implementing a computerized system that provides hospitals with fast and accurate analyses of patient opinions.

Bacteria, viruses, and parasites in an intermittent stream protected from and exposed to pasturing cattle: prevalence, densities, and quantitative microbial risk assessment.

Over 3500 individual water samples, for 131 sampling times, targeting waterborne pathogens/fecal indicator bacteria were collected during a 7-year period from 4 sites along an intermittent stream running through a small livestock pasture system with and without cattle access-to-stream restriction measures. The study assessed the impact of cattle pasturing/riparian zone protection on: pathogen (bacterial, viral, parasite) occurrence, concentrations of fecal indicators, and quantitative microbial risk assessments (QMRA) of the risk of Cryptosporidium, Giardia and Escherichia coli O157:H7 infection in humans. Methodologies were developed to compute QMRA mean risks on the basis of water samples exhibiting potentially human infectious Cryptosporidium and E. coli based on genotyping Crytosporidium, and E. coli O157:H7 presence/absence information paired with enumerated E. coli. All Giardia spp. were considered infectious. No significant pasturing treatment effects were observed among pathogens, with the exception of Campylobacter spp. and E. coli O157:H7. Campylobacter spp. prevalence significantly decreased downstream through pasture treatments and E. coli O157:H7 was observed in a few instances in the middle of the unrestricted pasture. Densities of total coliform, fecal coliform, and E. coli reduced significantly downstream in the restricted pasture system, but not in the unrestricted system. Seasonal and flow conditions were associated with greater indicator bacteria densities, especially in the summer. Norovirus GII was detected at rates of 7-22% of samples for all monitoring sites, and rotavirus in 0-7% of samples for all monitoring sites; pasture treatment trends were not evident, however. Seasonal and stream flow variables (and their interactions) were relatively more important than pasture treatments for initially stratifying pathogen occurrence and higher fecal indicator bacteria densities. Significant positive associations among fecal indicator bacteria and Campylobacter spp. detection were observed. For QMRA, adjusting for the proportion of Cryptosporidium spp. detected that are infectious for humans reduces downstream risk estimates by roughly one order of magnitude. Using QMRA in this manner provides a more refined estimate of beneficial management practice effects on pathogen exposure risks to humans.

The effect of pre-treatment and sonication of centrifugal ultrafiltration devices on virus recovery.

Enteric viruses such as norovirus (NV) and hepatitis A (HAV) are responsible for a large proportion of food and water-borne illnesses. Most human pathogenic enteric viruses cannot be cultured so they must be detected by molecular techniques. Male specific (F(+)) RNA coliphages, a potential surrogate for human enteric viruses, can be detected by culture and molecular assays. Numbers of viruses and F-RNA coliphages in contaminated food or water may be too low for direct detection. Ultrafiltration is a general concentration method for all virus types but there is little information on the recovery efficiency of F-RNA coliphages and enteric viruses. The recovery of F-RNA coliphage MS2 was only 25% by plaque assay in initial trials. The objective was to optimize the recovery of concentrated MS2 from Microsep 100K ultrafiltration devices. The mean recovery of MS2 increased significantly to 85% by plaque assay and 65% by real-time RT-PCR when ultrafiltration devices were treated with 1% BSA before concentration and then ultrasonicated after concentration. The method was validated with MS2, HAV, NV and feline calicivirus (FCV) in water and spinach eluate. The recovery of MS2, HAV and NV was significantly higher from concentrates obtained from water with treated devices than untreated devices but not significantly different for FCV or from spinach eluate. To our knowledge, this is the first study to use ultrasonication as a post-treatment step to increase recovery of viruses from ultrafiltration devices.

von Willebrand factor is not altered in azotemic dogs with prolonged bleeding time.

The effects of azotemia on von Willebrand factor (vWf) plasma concentration, structure, and function were studied by utilizing canine models for both uremic bleeding and type I vWf deficiency (vWd). Seventy-five percent to 80% renal mass reduction in eight mixed-breed dogs induced marked azotemia (blood urea nitrogen [BUN] 103 +/- 7 mg/dl [mean +/- SEM]; creatinine 5.8 +/- 1 mg/dl) and prolonged mean buccal mucosal bleeding time (BMBT) from 1.8 +/- 0.2 minutes to 7.0 +/- 0.4 minutes. The mean vWf plasma concentration increased from 0.88 +/- 0.11 U/ml to 1.26 +/- 0.14 U/ml. The pre- and postsurgical sodium dodecyl sulfate-agarose gel electrophoresis multimeric patterns were similar in all dogs. Administration of cryoprecipitate from pooled azotemic mixed-breed dog plasma to five Doberman pinschers with type I vWd increased the mean plasma vWf from 0.14 +/- 0.01 U/ml to 0.48 +/- 0.04 U/ml and decreased the BMBT from 7.1 +/- 0.6 minutes to 3.14 +/- 0.09 minutes. After renal mass reduction, five type I vWd Dobermans developed marked azotemia (BUN 79 +/- 8.6 mg/dl; creatinine 3.7 +/- 0.6 mg/dl) and prolonged BMBT (16.1 +/- 3.6 minutes). Findings in the eight azotemic mixed-breed dogs indicated that (1) vWf plasma levels were normal to increased in azotemic dogs; (2) vWf structure and multimeric distribution were not altered in canine azotemia; and (3) vWf was functional when placed in a non-azotemic environment. The prolongation of the BMBT in azotemic vWd dogs indicated that factors other than alteration of vWf function were responsible for the prolonged BMBT in canine azotemia.