A new method for simultaneous detection and discrimination of Bovine herpesvirus types 1 (BoHV-1) and 5 (BoHV-5) using real time PCR with high resolution melting (HRM) analysis.

Bovine herpesvirus types 1 (BoHV-1) and 5 (BoHV-5) are antigenically and genetically similar. The aim of this study was to develop a simple and reliable one-step real time PCR assay with high resolution melting (HRM) analysis for the simultaneous detection and differentiation of BoHV-1 and BoHV-5. Optimization of assay conditions was performed with DNA from reference strains. Then, DNA from field isolates, clinical samples and tissue samples of experimentally infected animals were studied by real time PCR-HRM. An efficient amplification of real time PCR products was obtained, and a clear melting curve and appropriate melting peaks for both viruses were achieved in the HRM curve analysis for BoHV type identification. BoHV was identified in all of the isolates and clinical samples, and BoHV types were properly differentiated. Furthermore, viral DNA was detected in 12/18 and 7/18 samples from BoHV-1- and BoHV-5-infected calves, respectively. Real time PCR-HRM achieved a higher sensitivity compared with virus isolation or conventional PCR. In this study, HRM was used as a novel procedure. This method provides rapid, sensitive, specific and simultaneous detection of bovine alpha-herpesviruses DNA. Thus, this technique is an excellent tool for diagnosis, research and epidemiological studies of these viruses in cattle.

The rate and pattern of urea infusion into the rumen of wethers alters nitrogen balance and plasma ammonia.

Changes in N balance, urinary excretion of purine derivative (PD), urea, creatinine and ammonia and plasma ammonia, glucose, urea, insulin and IGF-1 were examined in four wethers (37 ± 2.6 kg BW). The animals were fitted with permanent ruminal catheters, fed lucerne hay (9.4 MJ/day; 23 g N/day; 7 g soluble N/day, 6 equal meals/day) and treated with contrasting rates of urea infusion into the rumen: first, a continuous infusion (CT), at 3.2 mg urea-N/min for 10 days and then a discontinuous infusion (DT) at 156 mg urea-N/min for 4 min; in 6 daily doses with the meals for 7 days. N balance was calculated from pooled samples of faeces and urine. Jugular blood samples were collected before and 1.5 h after the morning meal (M1) on days CT10, DT2, DT4 and DT6. N retention decreased during DT (p = 0.01) due to a significant increase of N excretion in urine (4 g/day; p = 0.009) and faeces (1 g/day; p = 0.02). Dry matter (p < 0.001) and N digestibility in vivo (p = 0.01) decreased significantly during DT. Urinary urea and PD excretion were not altered by treatment. Significant linear (p = 0.004) and quadratic (p = 0.001) effects were observed for plasma ammonia in M1 (from 170 CT10 to 235 µm DT2 and returned to 120 µm DT6). No changes were observed in plasma glucose, urea, insulin and IGF-1. Results indicate that changes from CT to DT reduced N retention in sheep due to enhanced urinary N excretion, but it was not associated with changes in urinary urea or PD excretion; or plasma concentrations of insulin and IGF-1. As the dry matter (DM) an N digestibility could account a 0.23 of the decrease in N retention; the largest fraction of the reduction in N retention remained unexplained by the results.

Splanchnic net balance of oxygen and metabolites in response to a discontinuous mesenteric vein infusion of ammonium in sheep.

To simulate daily episodes of high absorption associated with the intake of diets with high N content, four wethers (42 ± 3.4 kg body weight), fitted with permanent catheters in the femoral artery and splanchnic vessels, were infused with 340 µmol into the mesenteric vein for 3 h, during the morning meal, over seven consecutive days. On the 7th day, mass transfers of , urea, glucose, lactate, ß-OH-butyrate and O2 were measured across portal-drained viscera (PDV), liver and splanchnic tissues during the last 90 min of the infusion. Measurements were repeated on the following day, at the same time, without the infusion. Plasma concentration in the portal vein (+332 µm; p = 0.006), portal absorption (+424 µmol/min; p < 0.001), liver uptake (+375 µmol/min; p = 0.003) and urea N production (+338 µmol/min; p = 0.059) were higher during infusion. Mass transfers of urea, glucose, lactate, ß-OH-butyrate and O2 across the PDV, and glucose, lactate, ß-OH-butyrate and O2 across the liver, were not altered by the infusion. Results suggest that a daily, discontinuous increase in portal flow during a meal stimulates liver removal and urea N production but does not significantly affect liver glucose production and O2 consumption in sheep.