Concentration and detection of caliciviruses in water samples by reverse transcription-PCR.

Human caliciviruses (HuCVs) cause waterborne outbreaks of gastroenteritis. Standard indicators of a safe water supply do not adequately predict contamination of water by viruses, including HuCVs. We developed a method to concentrate and detect HuCVs in water samples by using a cultivable primate calicivirus (Pan-1) as a model. Viable Pan-1 was seeded in different types of water and then filtered with a 1MDS filter, eluted with beef extract (BE), and reconcentrated by polyethylene glycol (PEG) precipitation. The viruses in the final samples were tested by plaque assay or by reverse transcription (RT)-PCR following extraction of the RNA with Trizol. Pan-1 was more sensitive to high-pH treatment than poliovirus was; a pH 9.0 BE solution was found to recover 35% more viable Pan-1 than a pH 9.5 BE solution recovered. Pan-1 was recovered from small volumes of deionized, finished, ground, and surface waters at efficiencies of 94, 73, 67, and 64%, respectively, when samples were assayed after elution without further concentration. When larger volumes of water (up to 40 liters) were tested after elution and concentration with PEG, 38, 19, and 14% of the seeded Pan-1 were recovered from finished, ground, and surface waters, respectively. The limit of detection of Pan-1 by RT-PCR was estimated to be 0.75 to 1.5 PFU in 40 liters of finished water. This method may be adapted for monitoring HuCVs in drinking water and other types of water for public health safety.

Repeatability and relationship with field mating performance of a serving capacity pen test in rams.

Two studies were conducted to analyse the repeatability of a serving capacity (SC) pen test and to evaluate the correlation of the results of such a test with field performance. The Laborde SC test, which measures SC by exposing an individual ram for 40min to two restrained non-oestrous ewes in a pen was used. In the first study, SC was measured on four and six occasions during the autumn of 1994 and 1995, respectively. The results indicated that this test is reliable in measuring SC of rams, since SC was repeatable within, and between, breeding season of consecutive years (p<0.05). In the second study conducted on two commercial farms, two groups of ewes synchronised with intravaginal sponges containing medroxyprogesterone acetate, were served by rams of high or low SC. On both the farms, the group of ewes served by rams of high SC had higher pregnancy rates than those served by low SC rams (p<0.05). The Laborde test proved to be a reliable test for the evaluation of SC in rams and that high SC rams had better field performance.

Detection of pathogen transmission in neonatal nurseries using DNA markers as surrogate indicators.

OBJECTIVE: Nosocomial infections are a major problem confronting neonatal intensive care units (NICUs). This study was conducted to determine if DNA markers designed from the cauliflower mosaic virus (CaMV 35S DNA) can serve as surrogate indicators of nosocomial pathogen transmission in NICUs. METHODS: Regions of cauliflower CaMV 35S promoter DNA were designed to serve as surrogate markers of microbial transmission pathways. Each of 6 pods within the NICU under study houses 8 newborn infants. DNA marker was placed on the telephone handle in only 1 of the 6 NICU pods (study pod). Bedside caregivers were blinded as to when placebo or marker were placed in the pod. Thirty-two samples were collected from predetermined sites within each pod at 0, 4, 8, 24, and 48 hours and 7 days after DNA placement. Similar sites were sampled in each of the 6 pods. Additional samples were collected concurrently from areas of the NICU segregated from direct patient care. Polymerase chain reactions were performed on collected samples, and products were analyzed by agarose gel electrophoresis. RESULTS: One thousand three hundred samples of the environment and hands of personnel were collected and analyzed. Within the study pod, 58% of sites tested positive for the DNA marker throughout all time points; positive sites peaked at 8 hours (78%) and declined to 23% positive at 7 days. The other 5 pods had a mean of 18% of sites positive throughout the 7 days and exhibited a similar decline throughout time. The most consistently positive sites within all pods were the blood gas analyzers, computer mice, telephone handles, medical charts, ventilator knobs, door handles, radiant warmer control buttons, patient monitors, and personnel hands. In areas outside the pods, the nurse's station, resident physician charting area, changing room, and staff break room had a mean of 50% positive sites throughout all time points. CONCLUSIONS: DNA markers proved useful as safe, surrogate indicators of microorganism transmission within and outside pods in the NICU. We speculate that utilization of these techniques in the hospital environment will provide important information about transmission of pathogens in the NICU, assist in developing and enforcing cleaning procedures, and permit testing of educational intervention programs targeting a decrease in nosocomial infections.nosocomial infection, neonatal intensive care, DNA marker, polymerase chain reaction, infection control.

Estimation of the shear stress on the surface of an aortic valve leaflet.

The limited durability of xenograft heart valves and the limited supply of allografts have sparked interest in tissue engineered replacement valves. A bioreactor for tissue engineered valves must operate at conditions that optimize the biosynthetic abilities of seeded cells while promoting their adherence to the leaflet matrix. An important parameter is shear stress, which is known to influence cellular behavior and may thus be crucial in bioreactor optimization. Therefore, an accurate estimate of the shear stress on the leaflet surface would not only improve our understanding of the mechanical environment of aortic valve leaflets, but it would also aid in bioreactor design. To estimate the shear stress on the leaflet surface, two-component laser-Doppler velocimetry measurements have been conducted inside a transparent polyurethane valve with a trileaflet structure similar to the native aortic valve. Steady flow rates of 7.5, 15.0, and 22.5 L/min were examined to cover the complete range possible during the cardiac cycle. The laminar shear stresses were calculated by linear regression of four axial velocity measurements near the surface of the leaflet. The maximum shear stress recorded was 79 dyne/cm2, in agreement with boundary layer theory and previous experimental and computational studies. This study has provided a range of shear stresses to be explored in bioreactor design and has defined a maximum shear stress at which cells must remain adherent upon a tissue engineered construct.

Colonization with enteroadherent, enterotoxigenic and enterohemorrhagic Escherichia coli among day-care center attendees in New Orleans, Louisiana.

BACKGROUND: E. coli strains producing specific virulence factors are frequently cited as causes of pediatric diarrhea in developing areas, although many well children from the same areas are colonized with these organisms. The role of these Escherichia coli in day-care center (DCC)-associated diarrhea in the United States has not been evaluated. METHODS: A cohort of 112 DCC attendees from 5 DCC in urban New Orleans were followed longitudinally with demographic data, biweekly routine stool samples and additional stool samples with episodes of diarrhea. E. coli isolates were routinely saved; diarrhea stool samples were tested to detect enterotoxigenic, enterohemorrhagic and enteroadherent strains; and the prevalence of these E. coli in children with and without diarrhea was investigated. RESULTS: During 225 child months of observation 21 episodes of diarrhea were documented and microbiologic data were available for 18. HEp-2 cell enteroadherent E. coli [mostly enteroaggregative (EAggEC) pattern] were identified in 6 of 18 (33.3%) diarrhea cases vs. 6 of 36 (16.6%) age-matched controls. However, the prevalence of EAggEC was very DCC-specific, with EAggEC found in 12 of 22 routine specimens from a DCC with recent EAggEC-related diarrhea vs. 0 of 11 routine specimens from age-matched children in another DCC without EAggEC-related diarrhea (P=0.002). Enterotoxigenic E. coli were uncommon in both ill and well children, and no enterohemorrhagic E. coli were detected. CONCLUSION: EAggEC were commonly isolated from children with and without diarrhea in certain DCC settings, although we cannot determine whether these strains caused diarrhea. Diarrhea-producing E. coli were not associated with diarrhea in this DCC population.

Reproductive seasonality of corriedale rams under extensive rearing conditions.

The objective of the present study was to describe seasonal changes in scrotal circumference (SC), live weight (LW), sperm morphology and plasma levels of testosterone (T) and thyroxine (T4) in young Corriedale rams reared under extensive conditions typical for the southern Latin American region. A total of 31 Corriedale rams, 11 months of age and with a LW of 36 +/- 1.1 kg and (SC) of 23.0 +/- 0.5 cm at the beginning of the experiment (September) were kept on natural pastures. At monthly intervals LW was recorded, animals were clinically examined, and SC was measured. None of the animals were used for breeding. Fifteen animals were randomly selected and bled once a month from January to December and plasma concentrations of T and T4 were determined. In addition, one semen sample was collected by electroejaculation and morphological studies were performed. The mean individual LW increase was 18 kg (50% of the initial LW) during the experiment (p < 0.01). LW decreased in autumn, with the nadir in late autumn. SC reached mean maximum levels in late summer (31.1 +/- 0.4 cm, p < 0.01), then decreased until the beginning of winter (26.3 +/- 0.4 cm, p < 0.01) and remained low until early spring (27.5 +/- 0.5 cm, p > 0.05) to increase again between mid-spring and the end of the experimental period the following summer (30.7 +/- 0.5 cm, p < 0.01). The mean SC in winter was 16% lower than that in late summer. Semen could be collected from the rams throughout the experiment. Frequencies of sperm head, mid-piece and total abnormalities showed monthly variation (p < 0.05), but tail abnormalities were not affected by month. Low abnormalities were found in autumn (9.4% +/- 2.2%). T was high during autumn (p < 0.01). Minimal T4 concentrations were observed during late summer and early autumn (p < 0.01) when T levels were high. Maximum T4 concentrations were registered in late autumn (p < 0.01); when SC was decreasing; in mid-spring (p < 0.01) one month after shearing and in early summer (p < 0.01). The results suggest that Corriedale rams under Uruguayan extensive management systems show a reproductive seasonality that, in general terms, coincides with photoperiodic variations.

Effect of fecal contamination on diarrheal illness rates in day-care centers.

Contact spread of enteropathogens in day-care centers is supported by the recovery (presence vs. absence) of fecal coliforms from hands and day-care center fomites. This prospective study was conducted to determine what, if any, quantitative measures of fecal coliforms predict the risk of diarrhea among day-care center attendees. Diarrheal illness without concomitant respiratory symptoms was monitored among 221 children aged < 3 years in 37 classrooms (24 day-care centers) through biweekly parental telephone interviews from October 1988 to May 1989 in Cumberland County, North Carolina. The risk of diarrhea was expressed as new episodes/classroom-fortnight. Contamination was expressed as the log10 fecal coliform count per unit of surface area, per toy, and per child and staff hands. Significant predictors of diarrheal risk were any hand contamination (p = 0.003) and the number of contaminated moist sites (hands, faucets, and sinks) (p = 0.006). After adjusting for the child/staff ratio using weighted multiple regression, the authors found that classrooms with either any hand contamination (p = 0.0015) or contamination on all moist sites (p = 0.015) had a significant twofold increased rate of diarrhea compared with classrooms without contamination. This was the first study to demonstrate an increased risk of diarrhea associated with fecal contamination and the frequent sink contamination in day-care centers.

Compositional and enzymatic characteristics of the Longissimus Dorsi muscle from large white, halothane-positive and halothane-negative pietrain, and hampshire pigs.

One-hundred-and-twenty-nine pigs from four genetic types-Large Whites, halothane-negative (HN) and halothane-positive (HP) Pietrains and Hampshires-were used to study some compositional and enzymatic muscle traits. Water, nitrogen, hydroxyproline and lipid contents, as well as lactate dehydrogenase and citrate synthase activities, were measured on all pigs, whereas glycogen phosphorylases (a and a + b), glycogen synthetase I + D and myosin isozyme pattern were determined on only fifty-six pigs. Hampshires were markedly different from the other genetic types with respect to the characteristics studied. Their Longissimus dorsi muscle contained less nitrogen (P < 0·0) and more lipid (P < 0·01) and presented higher hydroxyproline to nitrogen (P < 0·001) and water to nitrogen (P < 0·001) ratios, as well as noticeably higher citrate synthase (P < 0·01) and glycogen synthetase.

[Enzyme metabolic and contractile activities of 30 pig muscles. Relation with the final ph attained after death]. / Activités enzymatiques métaboliques et contractiles de 30 muscles du Porc. Relations avec le pH ultime atteint après la mort.

This paper presents a study of the metabolic and contractile types of 34 samples from 30 muscles in five crossbred Pietrain-Large White pigs 6 to 7 months old. The activity of the following enzymes was measured: glyceraldehyde-3-phosphate dehydrogenase (GAPDH), lactate dehydrogenase using high (LDH-h) or low (LDH-b) pyruvate concentrations in the reaction medium, citrate synthase (CS), and myofibrillar Ca-Mg activated ATPase. Haeminic iron and ultimate pH (pHu) were measured on the same samples. The results showed a negative, rather linear relationship between GAPDH, LDH and ATPase activities on the one hand and CS and haeminic iron on the other. Rather high correlations (r = 0.7 to 0.8) were observed between metabolic and contractile criteria and pHu; the red (slow) muscles had the highest pHu.