Cyprinid herpesvirus 3 as a potential biological control agent for carp (Cyprinus carpio) in Australia: susceptibility of non-target species.

Carp (Cyprinus carpio L.) is a pest species in Australian waterways, and cyprinid herpesvirus 3 (CyHV-3) is being considered as a potential biological control (biocontrol) agent. An important consideration for any such agent is its target specificity. In this study, the susceptibility to CyHV-3 of a range of non-target species (NTS) was tested. The NTS were as follows: 13 native Australian, and one introduced, fish species; a lamprey species; a crustacean; two native amphibian species (tadpole and mature stages); two native reptilian species; chickens; and laboratory mice. Animals were exposed to 100-1000 times the approximate minimum amount of CyHV-3 required to cause disease in carp by intraperitoneal and/or bath challenge, and then examined clinically each day over the course of 28 days post-challenge. There were no clinical signs, mortalities or histological evidence consistent with a viral infection in a wide taxonomic range of NTS. Furthermore, there was no molecular evidence of infection with CyHV-3, and, in particular, all RT-PCRs for viral mRNA were negative. As a consequence, the results encourage further investigation of CyHV-3 as a potential biocontrol agent that is specific for carp.

Effects of immunological castration and distiller's dried grains with solubles on carcass cutability and commercial bacon slicing yields of barrows slaughtered at two time points.

Male pigs were randomly assigned to a castration method at birth and allotted to 48 pens (28 pigs/pen). Physically castrated (PC) barrows were castrated at 2 d of age; immunologically castrated (IC) barrows were administered Improvest (GnRF analog diphtheria toxoid conjugate; Zoetis, Kalamazoo, MI) at 16 and 20 wk of age. Distiller's dried grains with solubles (DDGS) feeding strategies included either 0% DDGS (control), 30% DDGS (30% DDGS) fed from 6 wk of age to slaughter, or 30% DDGS fed from 6 wk of age to second dose of Improvest and then fed 0% DDGS until slaughter (withdrawal). Four barrows closest to the median pen weight at 4.5 wk after second dose were selected for evaluation; two were randomly selected and slaughtered at 5 wk and the other two at 7 wk after second dose. Data from each slaughter time were analyzed independently as a 2 × 3 factorial design with pen as the experimental unit. At 5 wk after second dose, bone-in lean cutting yields were 2.63% units greater (P < 0.01) in IC when compared to PC. Bellies were thicker (P < 0.01) and tended to have greater belly flop distances (P = 0.07) in PC compared to IC, however iodine values (IV) were not altered (P = 0.84). Carcass traits (P ≥ 0.10), cutting yields (P ≥ 0.43), and fresh belly characteristics (P ≥ 0.08) were minimally affected by DDGS feeding strategy. Bacon slicing yields (percentage of green weight) were 6.10% units less (P < 0.01) in IC compared with PC. At 7 wk after second dose, bone-in lean cutting yields were 1.57% units greater (P = 0.03) in IC compared with PC. Distiller's grains feeding strategy had no effect (P ≥ 0.83) on boneless carcass cutting yields in IC; while in PC, these yields were 2.32% units less (P < 0.02) in control-fed barrows when compared to other feeding strategies (castration method × feeding strategy; P = 0.03). Bellies from PC tended to be thicker (P = 0.07) and have similar flop distances (P = 0.44) and IV (P = 0.54) when compared with IC. Iodine value was greater (P = 0.03) in 30% DDGS-fed barrows compared with control-fed barrows. Bacon slicing yields (percentage of green weight) were 4.27% units less (P = 0.05) in IC compared with PC. These data suggested that while bacon slicing yield was reduced in IC barrows fed control and 30% DDGS compared with PC barrow counterparts, withdrawal of DDGS improved bacon slicing yields of IC barrows.

The effects of immunological castration and corn dried distillers grains with solubles withdrawal on growth performance, carcass characteristics, fatty acid analysis, and iodine value of pork fat depots.

A total of 1,360 pigs were used in a 125-d study to determine the effects of corn dried distillers grains with solubles (DDGS) withdrawal after immunological castration (Improvest, Zoetis, Kalamazoo, MI) on growth performance and carcass fat quality of pigs. Pens of male pigs (initially 24 kg) were randomly allotted by BW and castration method (physically castrated [PC] or immunologically castrated [IC] barrows) to 1 of 3 diets with 8 replications per treatment and 27 to 29 pigs per pen. Treatments were arranged in a 2 × 3 factorial with main effects of castration method and diet (0% DDGS throughout, 30% DDGS throughout, or 30% DDGS through d 75 then no DDGS to d 125). Intact males were injected with Improvest on d 39 and 74 (IC). No castration method × diet interactions (P > 0.12) were observed for growth performance. Before the second Improvest injection (d 0 to 74), PC barrows had increased (P < 0.05) ADFI but were less efficient (P < 0.05) than intact males. After the second Improvest injection until the first marketing event (d 74 to 107), IC barrows had improved (P < 0.05) ADG and G:F compared with PC barrows. From d 0 to 107, IC barrows had improved (P < 0.05) ADG, G:F, and lower ADFI than PC barrows. The inclusion of 30% DDGS decreased (P < 0.05) G:F compared with pigs fed the control diet. For the period after the second Improvest injection (d 74 to 125), IC barrows had increased (P < 0.05) ADG, ADFI, and G:F compared with PC barrows. Overall (d 0 to 125), IC barrows had improved (P < 0.05) ADG and G:F and lower ADFI than PC barrows. The inclusion of 30% DDGS decreased (P < 0.05) G:F. Carcass yield was lower (P < 0.05) for IC than PC barrows. Pigs fed 30% DDGS throughout had decreased (P < 0.05) carcass yield; however, withdrawing DDGS from the diet on d 74 was effective at fully recovering the yield loss. Carcass fat iodine values (IV) were consistently higher (P < 0.05), regardless of fat depot or harvest time when 30% DDGS were included in the diet. Multiple 2-way interactions (P < 0.05) were detected between castration method, DDGS, depot, and time. Interactions were a result of fatty acid profiles changing more rapidly in backfat and belly fat than in jowl fat from d 107 to 125 and more dramatically in IC than PC barrows in the same period. This improvement from d 107 to 125 could be caused by the dilution of unsaturated fatty acids, specifically C18:2 and C18:3, due to rapid deposition of fat from de novo synthesis in IC barrows.

Effects of lowering dietary fiber before marketing on finishing pig growth performance, carcass characteristics, carcass fat quality, and intestinal weights.

A total of 264 pigs (initially 41.0 kg BW) were used in a 90-d study to determine the effects of lowering dietary fiber before market on pigs fed high dietary fiber [provided by wheat middlings (midds) and distillers dried grains with solubles (DDGS)] on growth performance, carcass characteristics, carcass fat quality, and intestinal weights of growing-finishing pigs. Pens of pigs were randomly allotted by initial BW and sex to 1 of 6 treatments with 6 replications per treatment and 7 or 8 pigs per pen. A positive control (corn-soybean meal-based) diet containing no DDGS or midds (9.3% NDF) and a negative control diet with 30% DDGS and 19% midds (19% NDF) were fed throughout the entire trial (d 0 to 90). The other 4 treatments were arranged in a 2 × 2 factorial with the main effects of length of fiber reduction (23 or 47 d before marketing) and fiber level fed during the reduction period (low or medium). Pigs on these treatments were fed the negative control before the reduction treatment. The medium-fiber diet contained 15% DDGS and 9.5% midds (14.2% NDF) with the low-fiber diet was the positive control diet. Increasing the feeding duration of the low-fiber diets lowered overall ADFI (linear, P = 0.03) and improved G:F (linear, P < 0.01). Lowering the fiber level for the last 23 d did not influence growth performance; however, lowering the fiber level improved carcass yield (P = 0.002), with a greater response (P < 0.001) when the low-fiber diet was fed for 23 d. Jowl fat iodine value (IV) decreased when the longer lower fiber diets were fed (linear, P < 0.01) and was lower (P < 0.001) for pigs fed the low-fiber diet during the fiber reduction period than pigs fed the medium-fiber diet during the same time period; however, increasing the time lower fiber diets were fed from 23 to 47 d further reduced (P < 0.01) jowl IV. Increasing the duration that the control diet was fed by increasing the reduction time from 23 to 47 d increased (P < 0.01) backfat depth. Reducing the fiber level decreased full large intestine weight (linear, P = 0.005) with a greater response (P = 0.04) when the low-fiber diet was fed during the reduction period instead of the medium-fiber diet. In summary, lowering the fiber level before marketing can improve G:F, carcass yield, carcass IV, and reduce large intestine weight; however, the optimal duration of the fiber reduction period depends on the targeted response criteria.

In vitro characteristics of tobramycin aerosol from ultrasonic and jet nebulizers.

STUDY OBJECTIVE: To compare the in vitro performance of an ultrasonic nebulizer and a jet nebulizer in producing a respirable aerosol of tobramycin solution for injection. DESIGN: In vitro observational study DEVICES: Ultrasonic and jet nebulizers. INTERVENTION: Output was determined by measuring the difference in nebulizer weight before and after nebulizing 3 ml of tobramycin injection solution. Mass median aerodynamic diameter (MMAD) and respirable mass were determined by sampling tobramycin aerosol into a cascade impactor. MEASUREMENTS AND MAIN RESULTS: Mean (SD) output was 1.14 (0.09) ml/minute for the ultrasonic nebulizer and 0.64 (0.08) ml/minute (p<0.001) for the jet nebulizer. Mean MMAD for the jet nebulizer (2.31 [0.10] microm) was less than that of the ultrasonic nebulizer (2.81 [0.17] microm, p<0.001). The majority of tobramycin aerosol produced was in the respirable range for both the ultrasonic (65.1% [4.10%]) and jet (60.6% [0.73%], p=0.008) nebulizers. CONCLUSION: Despite small, clinically unimportant differences in aerosol size and respirable fraction, either device would be acceptable to administer tobramycin injection solution.

Pulmonary function response to EDTA, an additive in nebulized bronchodilators.

BACKGROUND: Some nebulized bronchodilator solutions contain additives, such as EDTA, benzalkonium chloride (BAC), or both. OBJECTIVE: Although BAC-induced bronchoconstriction has been well documented in patients with asthma, there is no information on the effects of EDTA on FEV(1) when inhaled in the amounts that would be administered during emergency department treatment of asthma. METHODS: Eighteen subjects with stable asthma and airway responsiveness to methacholine were randomly assigned to inhale up to four 600-microg nebulized doses of EDTA, BAC (positive control), and normal saline (placebo) in a double-blind crossover manner on separate days. FEV(1) was measured 15 minutes after each dose. Treatments were repeated every 20 minutes until FEV(1) decreased by 20% or greater or a maximum of 4 doses were administered. RESULTS: Mean +/- SD maximum percent decrease in FEV(1) was 1.8% +/- 5.8% after EDTA, 16.6% +/- 13.9% after BAC, and 3.6% +/- 8.2% after placebo (P <.001); there was no significant difference between EDTA and placebo. CONCLUSION: The amount of EDTA contained in maximum recommended doses of nebulized bronchodilators does not induce bronchospasm. In contrast, BAC induces clinically important bronchospasm, which could decrease the efficacy of a bronchodilator during an emergency.

The M3 muscarinic acetylcholine receptor expressed in HEK-293 cells signals to phospholipase D via G12 but not Gq-type G proteins: regulators of G proteins as tools to dissect pertussis toxin-resistant G proteins in receptor-effector coupling.

The M(3) muscarinic acetylcholine receptor (mAChR) expressed in HEK-293 cells couples to G(q) and G(12) proteins and stimulates phospholipase C (PLC) and phospholipase D (PLD) in a pertussis toxin-insensitive manner. To determine the type of G protein mediating M(3) mAChR-PLD coupling in comparison to M(3) mAChR-PLC coupling, we expressed various Galpha proteins and regulators of the G protein signaling (RGS), which act as GTPase-activating proteins for G(q)- or G(12)-type G proteins. PLD stimulation by the M(3) mAChR was enhanced by the overexpression of Galpha(12) and Galpha(13), whereas the overexpression of Galpha(q) strongly increased PLC activity without affecting PLD activity. Expression of the RGS homology domain of Lsc, which acts specifically on Galpha(12) and Galpha(13), blunted the M(3) mAChR-induced PLD stimulation without affecting PLC stimulation. On the other hand, overexpression of RGS4, which acts on Galpha(q)- but not Galpha(12)-type G proteins, suppressed the M(3) mAChR-induced PLC stimulation without altering PLD stimulation. We conclude that the M(3) mAChR in HEK-293 cells apparently signals to PLD via G(12)- but not G(q)-type G proteins and that G protein subtype-selective RGS proteins can be used as powerful tools to dissect the pertussis toxin-resistant G proteins and their role in receptor-effector coupling.

Levalbuterol nebulizer solution: is it worth five times the cost of albuterol?

Albuterol is a 50:50 mixture of R-albuterol, the active enantiomer, and S-albuterol, which appears to be inactive in humans. The Food and Drug Administration recently approved levalbuterol, the pure R-isomer, as a preservative-free nebulizer solution. Published studies indicate that it is neither safer nor more effective than an equimolar dose of racemic albuterol (levalbuterol 1.25 mg = albuterol 2.5 mg). However, these studies were conducted in patients with stable asthma (at the top of the dose-response curve), whereas a nebulized bronchodilator most likely would be used by patients with an acute exacerbation. Because such patients, in the hospital setting, often require higher doses of albuterol, the manufacturer's recommended dose of levalbuterol is likely to be too low for rescue therapy. Levalbuterol may cost as much as 5 times more than racemic albuterol, depending on purchase method. We conclude that levalbuterol offers no advantage over albuterol but is likely to be more costly.

Bronchoconstrictor additives in bronchodilator solutions.

Nebulized bronchodilator solutions are available in the United States as both nonsterile and sterile-filled products. Sulfites, benzalkonium chloride (BAC), or chlorobutanol are added to nonsterile products to prevent bacterial growth, but there have been reports of contaminated solutions containing preservatives. Ethylenediamine tetraacetic acid (EDTA) is added to some products to prevent discoloration of the solution. With the exception of chlorobutanol, all of these additives are capable of inducing bronchospasm in a concentration-dependent manner. However, it is rarely apparent to the patient or health care provider that the additive diminishes the bronchodilator effects. Older products (eg, isoproterenol and isoetharine) contain enough sulfites to produce bronchospasm in most patients with asthma, even in those without a prior history of sulfite sensitivity. Bronchoconstriction from inhaled BAC is cumulative, prolonged, and correlates directly with basal airway responsiveness. The multidose dropper bottle of albuterol contains 50 microg BAC/dose, which is below the threshold for bronchoconstriction whereas the screwcap unit-dose vial contains 300 microg/dose, which is above the threshold for many patients. If the screwcap product is used in the emergency department, a patient could receive as much as 1800 microg of BAC in the first hour. Three sterile-filled unit dose albuterol products contain no additives, whereas a fourth, (manufactured by Dey Laboratories) contains 300 microg of EDTA, which is also below the threshold dose for bronchoconstriction. Only additive-free sterile solutions should be used for hourly or continuous nebulization of albuterol. The multidose dropper bottle or the Dey product can be used when the interval between doses is longer, whereas the screwcap product should not be used for acute therapy. Ipratropium is available only as a sterile, additive-free unit-dose vial, as is levalbuterol.

The ADP-ribosylation factor (ARF)-related GTPase ARF-related protein binds to the ARF-specific guanine nucleotide exchange factor cytohesin and inhibits the ARF-dependent activation of phospholipase D.

ADP-ribosylation factor-related protein (ARP) is a membrane-associated GTPase with remote similarity to the family of ADP-ribosylation factors (ARF). In a yeast two-hybrid screen designed to identify proteins interacting with ARP, we isolated a partial cDNA of the ARF-specific guanine nucleotide exchange factor mSec7-1/cytohesin encoding its N terminus and most of the Sec7 domain (codons 1-200). ARP and ARP-Q79L (GTPase-negative ARP) exhibited a higher affinity to mSec7-1-(1-200) than ARP-T31N (nucleotide exchange-defective ARP) in the two-hybrid assay. Similarly, full-length [35S]mSec7-1/cytohesin was specifically adsorbed to glutathione-Sepharose loaded with glutathione S-transferase (GST)-ARP-Q79L, GST-ARP, or GST-ARP-T31N, the latter exhibiting the lowest binding affinity. Overexpression of ARP-Q79L, but not of ARP-T31N, in COS-7 cells reduced the fluorescence from co-expressed green fluorescent protein fused with mSec7-1/cytohesin or mSec7-2/ARNO in plasma membranes as detected by deconvolution microscopy. Recombinant ARP and ARP-Q79L, but not ARP-T31N, inhibited the phospholipase D (PLD) activity stimulated by mSec7-2/ARNO and ARF in a system of isolated membranes. Furthermore, transfection of HEK-293 cells with ARP or ARP-Q79L, but not ARP-T31N, inhibited the muscarinic acetylcholine receptor-3 induced PLD stimulation and translocation of ARF from cytosol to membranes. These data suggest that the GTP-bound form of ARP specifically binds mSec7-1/cytohesin, and that ARP may be involved in a pathway inhibiting the ARF-controlled activity of PLD.

Evaluation of a noninstrumented disposable method for quantifying serum theophylline concentrations.

STUDY OBJECTIVE: To compare the performance of a new point-of-care theophylline assay (AccuMeter) with that of a standard laboratory assay (TDx), and another point-of-care method (AccuLevel). DESIGN: Prospective evaluation of consecutive patients receiving theophylline. SETTING: University-based, ambulatory, allergy-pulmonary clinic. PATIENTS: Forty subjects receiving maintenance theophylline therapy for asthma. INTERVENTIONS: Theophylline concentrations obtained from AccuMeter, TDx, and AccuLevel were compared. MEASUREMENTS AND MAIN RESULTS: The error, or difference, between TDx and AccuMeter results in 40 subjects on maintenance theophylline described accuracy. Mean error, an estimate of bias, was 1.1 (95% CI 0.72-1.5), 0.67 (0.34-1.0), and 0.98 (0.79-1.2) microg/ml for AccuMeter capillary, serum, and heparinized blood samples. Square root of the mean squared error, an estimate of precision, was 1.6 (1.2-2.0), 1.22 (0.90-1.5), and 1.14 (0.96-1.3) microg/ml for AccuMeter capillary, serum, and heparinized samples. Difference between AccuMeter and AccuLevel ME, an estimate of relative bias, was 0.59 (0.04-1.1) microg/ml. The difference in mean squared errors, an estimate of relative precision, was 0.86 (-0.54-2.3) microg/ml. CONCLUSIONS: AccuMeter demonstrated good precision and minimal bias compared with TDx and AccuLevel . Method of sample collection had no effect on its accuracy.

Apparent decrease in population clearance of theophylline: implications for dosage.

BACKGROUND: Having observed in recent years that the theophylline dose requirements needed to attain peak serum concentrations of 10 to 20 micrograms/ml infrequently reached previously described mean values, we hypothesized that a downward shift in the range of dose requirements had occurred among patients with asthma. STUDY DESIGN: We examined dosage requirements needed to attain peak serum concentrations of 10 to 20 micrograms/ml in all patients with chronic asthma treated with theophylline by the Pediatric Allergy and Pulmonary Clinic at the University of Iowa from 1990 to 1994 (n = 300) and at the Pediatric Pulmonary Clinic at the University of Florida from 1992 to 1995 (n = 93). We then compared these doses to previous dose requirements from 1978 to 1983 determined in the same manner. RESULTS: Despite similar mean peak serum concentrations during both time periods (14 micrograms/ml), mean theophylline dosage requirements during the period of this study were approximately 25% lower among all age groups than those previously observed (p < 0.001). There were no significant differences in mean dosage requirements between the Iowa and Florida patients in any age group examined. CONCLUSIONS: Theophylline dose requirements needed to attain serum concentrations of 10 to 20 micrograms/ml have decreased significantly from those on which current dosing recommendations are based. This suggests a decrease in mean clearance of the population.