RESUMEN

Animal welfare and economic implications of infectious diseases in cattle demand an efficient surveillance as the foundation for control and eradication programmes. Bovine respiratory syncytial virus (BRSV), Parainfluenza virus type 3 (PI3V), Bovine herpes virus-1 (BoHV-1), Bovine viral diarrhoea virus (BVDV), and Enzootic bovine leukosis virus (EBLV) cause common and often underdiagnosed diseases in cattle that are endemic in most countries [1]. A hallmark of individual exposure to a viral pathogen is the presence of antibodies directed towards that virus. The aim of this study was to develop and validate a pentaplex assay to simultaneously detect and quantify antibodies against BRSV, PI3V, BoHV-1, BVDV and EBLV in serum, as an efficient tool to yield epidemiological data. Monoplex assays were initially developed using either complete BRSV or BoHV-1 viral lysates, or recombinant proteins for BVDV, EBLV or PI3V as capture antigens. In addition, 125 serum samples from unvaccinated cattle, which were classified as positive or negative for each of the viruses by commercial ELISA kits, were used for validation. Conditions established for the Luminex monoplex assays were adopted for the pentaplex assay. The accuracy, determined by the area under the ROC curve, was greater than 0.97, and assay diagnostic sensitivities and specificities were over 95 and 90%, respectively, for all antigens. Intra (r) and interassay (R) coefficients of variation were under 10 and 20 %, respectively. Selectivity towards target viruses was shown by binding inhibition assays where unbound viruses reduced fluorescence intensities. Diagnostic agreement for samples analysed simultaneously in the monoplex and multiplex assays was almost perfect. In conclusion, a highly sensitive pentaplex assay was validated for the simultaneous identification of antibodies directed against BVDV, BoHV-1, PI3V, BRSV and EBLV in serum. The developed pentaplex assay complies with performance characteristics established by international guidelines for diagnostic tests and may be used as a tool for the implementation of epidemiological surveillance.

RESUMEN

The frequency of multiple fetuses has increased in human pregnancies due to assisted reproductive technologies. This translates into a greater proportion of premature and low-birth weight infants in the United States and worldwide. In addition, improvements in sheep breeding have resulted in new breeds with increased litter size but reduced fetal survival and birth weight. Currently, there are no treatments for preventing fetal growth restriction in humans or sheep (an established model for studying human fetal physiology) carrying multiple fetuses. In this work, Booroola Rambouillet ewes (FecB+/-) with 2-4 fetuses were fed a diet providing 100% of NRC-recommended nutrient requirements. Between d 100 and 121 of gestation, ewes received an i.v. bolus injection of either saline solution or 345 µmol arginine-HCl/kg body weight 3 times daily. The arginine treatment reduced (P < 0.05) the percentage of lambs born dead by 23% while increasing (P = 0.05) the percentage of lambs born alive by 59%. The i.v. administration of arginine enhanced (P < 0.05) the birth weights of quadruplets by 23% without affecting maternal body weight. The improved pregnancy outcome was associated with an increase in maternal plasma concentrations of arginine, ornithine, cysteine, and proline, as well as a decrease in circulating levels of ammonia and ß-hydroxybutyrate. These novel results indicate that parenteral administration of arginine to prolific ewes ameliorated fetal mortality and growth retardation. Our findings provide support for experiments to assess the clinical use of arginine to enhance fetal growth and survival in women gestating multiple fetuses.

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RESUMEN

Intrauterine growth restriction (IUGR) is a major health problem worldwide that currently lacks an effective therapeutic solution. This study was conducted with an ovine IUGR model to test the hypothesis that parenteral administration of l-arginine (Arg) is effective in enhancing fetal growth. Beginning on d 28 of gestation, ewes were fed a diet providing 100% (control-fed) or 50% (underfed) of NRC-recommended nutrient requirements. Between d 60 of gestation and parturition, underfed ewes received i.v. infusions of saline or 155 micromol Arg-HCl/kg body weight 3 times daily, whereas control-fed ewes received only saline. The birth weights of lambs from saline-infused underfed ewes were 23% lower (P < 0.01) than those of lambs from control-fed dams. Administration of Arg to underfed ewes increased (P < 0.01) concentrations of Arg (69%), ornithine (55%), proline (29%), methionine (37%), leucine (36%), isoleucine (35%), cysteine (19%), and FFA (43%) in maternal serum, decreased maternal circulating levels of ammonia (18%) and triglycerides (32%), and enhanced birth weights of lambs by 21% compared with saline-infused underfed ewes. There was no difference in birth weights of lambs between the control-fed and the Arg-infused underfed ewes. These novel results indicate that parenteral administration of Arg to underfed ewes prevented fetal growth restriction and provide support for its clinical use to ameliorate IUGR in humans. The findings also lay a new framework for studying cellular and molecular mechanisms responsible for the beneficial effects of Arg in regulating conceptus growth and development.

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L-arginine administration may be useful for the treatment of intrauterine growth restriction, but concerns remain about effective precursors for administration into pregnant dams. Therefore, we used an ovine model to test the hypothesis that infusion of L-citrulline into the maternal circulation increases L-arginine availability to the fetus. On d 135 +/- 1 of gestation, ewes received an i.v. bolus dose of L-citrulline (155 micromol/kg body weight) or the same dose of L-arginine-HCl. Maternal and fetal arterial blood samples were obtained simultaneously at -120, -60, 0, 5, 15, 30, 60, 120, 180, and 240 min relative to the time of amino acid administration. Concentrations of arginine in maternal plasma increased to peak values within 5 min after its injection in ewes and declined rapidly thereafter, whereas concentrations of arginine in fetal plasma increased between 15 and 30 min and returned to baseline values by 60 min. In contrast, administration of citrulline increased concentrations of citrulline and arginine in maternal and fetal plasma between 5 and 60 min and values remained elevated thereafter. The differential pharmacokinetics for arginine compared with citrulline infusion was consistent with the observation that the half-life of citrulline was twice that of arginine in ewes. We conclude that i.v. administration of citrulline is more effective than arginine in sustaining high concentrations of arginine in the maternal and fetal circulations of pregnant ewes. These novel findings provide support for studies of the clinical use of arginine and citrulline as therapeutic means to prevent or ameliorate fetal growth retardation in mammals.

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Anticipating the future use of arginine to enhance fetal and neonatal growth as well as to treat diabetes and obesity, we performed studies in pigs, rats, and sheep to determine the pharmacokinetics of orally or i.v. administered arginine and the safety of its chronic supplementation. Our results indicate that all 3 species rapidly catabolized the supplemental arginine. The elevated circulating concentrations of arginine generally returned to baseline levels within 4-5 h after administration, with the rates varying with the age and physiological status of the animals. The clearance of arginine was greater in pregnant than in nonpregnant animals, in young than in adult animals, in lean than in obese animals, and in type-1 diabetic than in nondiabetic animals. I.v. administration of arginine-HCl to pregnant ewes (at least 0.081 g arginine.kg body weight-1.d-1) did not result in any undesirable treatment-related effect. Neonatal pigs, growing-finishing pigs, pregnant pigs, and adult rats tolerated large amounts of chronic supplemental arginine (e.g. 0.62, 0.32, 0.21, and 2.14 g.kg body weight-1.d-1, respectively) administered via enteral diets without the appearance of any adverse effect. On the basis of the comparative studies and a consideration of species differences in food intake per kilogram body weight, we estimate that a 70-kg human subject should be able to tolerate long-term parenteral and enteral supplemental doses of 6 and 15 g/d arginine, respectively, in addition to a basal amount of arginine (4-6 g/d) from regular diets.

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