High HCV subtype heterogeneity in a chronically infected general population revealed by high-resolution hepatitis C virus subtyping.

OBJECTIVES: This study aimed to characterize the chronically infected general hepatitis C virus (HCV) population in Barcelona using a highly sensitive subtyping method that can identify the 67 recognized HCV subtypes and diagnose mixed infection by various genotypes/subtypes in a single individual. The resulting information has implications for selecting optimal direct-acting antiviral (DAA) treatment for each patient and establishing public healthcare policies in our setting. METHODS: Consecutive HCV patients (treatment-naïve or interferon-based failures) attending Vall d'Hebron Hospital outpatient clinics from February 2015 to May 2016 (N=1473) were included in the study. Patient samples were characterized using HCV subtyping by next-generation ultra-deep pyrosequencing. RESULTS: The following genotypes (G) were found: G1 (1126/1473 (76.4%)), G4 (145/1473 (9.8%)), G3 (135/1473 (9.2%)), G2 (51/1473 (3.5%)), and G5 (1/1473 (0.1%)). Twenty-two subtypes were seen: 1b (790/1473 (53.6%)), 1a (332/1473 (22.5%)), 3a (133/1473 (9.0%)), 4d (105/1473 (7.1%)), 4a (29/1473 (2.0%)), and 2c (25/1473 (1.7%)), with 16 low-prevalence subtypes accounting for the remaining 3.0% (44/1473). There was a worrisome 1.0% (15/1473) of mixed infections. G2 (51/1473 (3.5%)) showed a high level of heterogeneity. Analyses by age groups showed a predominance of G1b over G1a (428/506 (84.6%) vs. 24/506 (4.7%)) in patients born before 1950 (N=506/1473), and similar percentages of these subtypes in those born between 1951 and 1975 (N=834/1473) (315/834, 37.8% vs. 266/834, 31.9%) and after 1976 (N=133/1473) (47/133, 35.3% vs. 42/133, 31.6%). CONCLUSIONS: Subtype distribution showed a higher level of heterogeneity than was expected, particularly for G2. Prevalence of mixed infections was around 1%. HCV subtype distribution related to patient age group suggested that patients born from 1936 to 1975 in our setting should undergo screening for the infection. Next-generation sequencing enabled better classification of candidates for DAA-based treatment.

Short- and long-term effects of forage supplementation of calves during the preweaning period on performance, reproduction, and milk yield at first lactation.

Sixty female Holstein calves [body weight (BW)=39.5±3.76kg] were fed a ground starter concentrate [19% crude protein, 19% neutral detergent fiber (NDF)] during the preweaning period. Furthermore, oats hay (68% NDF) was supplemented only during the postweaning period (CON) or during both pre- and postweaning periods (OH) to evaluate performance until first breeding, diet digestibility after weaning, reproductive performance, and milk yield at first lactation. Calves were individually housed and bedded with wood shavings. All calves were offered 6 L/d of milk replacer (MR) at 12% dry matter (DM) in 2 feedings until 28d of age, 3 L/d of MR at 12% DM in 2 feedings from 29 to 44d of age calves, and 1.5 L of MR at 12% DM in 1 feeding from 45 to 51d of age. Animals were weaned at 52d of age. Starter concentrate and forage intake were recorded daily and BW weekly until 65d of age. Two weeks after weaning, total-tract apparent digestibility was determined in 6 calves per treatment. Heifer BW was recorded at 10 mo of age. Breeding and milk yield at first lactation were also recorded. Starter concentrate intake was greater in OH compared with CON animals during the preweaning period. As a result, calves in the OH treatment had greater average daily gain (ADG) than CON animals during the preweaning period. After weaning, OH calves consumed more forage than CON animals, but we found no differences between treatments in ADG and starter concentrate intake. Similarly, total-tract apparent digestibility did not differ between treatments, and BW and ADG from 2wk after weaning to 10mo of age did not differ between treatments. Moreover, no differences in reproductive performance [age at first artificial insemination (AI), age at fertile insemination, conception rate at first AI, and number of AI] or milk yield at first lactation were observed between treatments, although a positive relationship between growth rate early in life and future energy-corrected milk yield was found. We conclude that offering forage to young calves early in life allows improvements in growth before weaning and could help in the transition to mixed diets, but the improvement in growth achieved early in life was not maintained at 10mo of age.

Interaction between the physical form of the starter feed and straw provision on growth performance of Holstein calves.

Two experiments were conducted to assess the effect of physical form of a starter feed with or without straw supplementation on growth performance of Holstein calves. In experiment 1, a total of 32 calves were randomly assigned at 7 d of age to texturized starter feed (containing rolled barley, corn, and oats) without straw, texturized starter feed with chopped straw, and pelleted starter feed with chopped straw. All calves were offered 4 L of pasteurized whole milk twice daily from 7 to 35 d of age, 2 L of milk twice daily from 36 to 42 d of age, and 2 L of milk from 43 to 49 d of age. Animals were weaned at 50 d of age, and the study finished when calves were 63 d old. In experiment 2, a total of 60 calves (8 d of age) were randomly assigned to texturized starter feed (containing whole corn) without straw, pelleted starter feed without straw, and pelleted starter feed with chopped straw. All calves were offered the same milk replacer (MR; 23% crude protein and 19.5 fat) at 11% dry matter concentration, 4 L/d of MR until 14 d of age, 6 L/d of MR from 14 to 37 d, 3 L/d of MR from 38 to 44 d, and 1.5 L/d of MR from 45 to 52 d of age. The experiment finished when calves were 58 d old (1 wk after weaning). Rumen liquid pH was measured after weaning. In both studies, calves were individually housed in pens on sawdust bedding and starter feed and chopped straw were offered free choice in separate buckets. In experiment 1, starter feed and straw intake and growth did not differ among treatments. However, calves receiving straw showed a greater rumen pH compared with those not receiving straw. In experiment 2, pelleted started feed supplemented with straw fostered an increase in solid feed intake (as percentage of body weight) compared with a pelleted or texturized starter feed without straw supplementation. However, calves that received the texturized starter feed containing whole corn had rumen pH similar to those fed a pelleted starter feed with straw. Feeding a texturized starter feed containing rolled barley, corn, and oats (with or without straw provision) was not able to maintain rumen pH or promote growth and intake compared with offering a pelleted starter feed with chopped straw. However, when whole corn was used in the texturized starter feed, rumen pH was equivalent to that obtained with a pelleted starter feed and straw supplementation.

Effects of particle size and moisture levels in mixed rations on the feeding behavior of dairy heifers.

Two experiments on replacement heifers (175±12 days of age) assessed the effects of forage particle length and moisture on feeding behavior. Both experiments used a replicated 3×3 Latin square design, with nine heifers per replication and three periods of 9 days each. Each group of nine heifers was housed in one pen with access to three electronic feed bins. In Experiment 1, hay chopped at different lengths was incorporated into three total mixed rations (TMR) all having the same ingredient and nutrient composition but differing in the percentage of long particles (>19 mm): 60% (Short), 64% (Medium) and 72% (Long). In Experiment 2, heifers were fed a TMR with the same ingredient and nutrient composition but differing in moisture content: 65% DM (Dry), 50% DM (Moderate), and 35% DM (Wet). In both experiments, feeding behavior during the last 5 days of each period was analyzed using a mixed model accounting for the fixed effects of treatment and period, and the random effects of replication and animal. In Experiment 1, dry matter intake (DMI) and eating rate (DMI/min) tended to increase, whereas daily eating time decreased as the feed particle size decreased. Heifers fed the Long diet selected in favor of long particles (>19 mm) and against Short (1.18 to 8 mm) and fine (<1.18 mm) particles; heifers fed the Short diet selected against long particles and in favor of short and fine particles. Heifers fed the Medium diet showed a preference for medium particles with no preference for the other particle sizes. In Experiment 2, heifers fed the Dry diet tended to consume more feed than those fed the Moderate and Wet diets, with no differences in feeding behavior or sorting activity. In conclusion the Medium diet minimized sorting without reducing eating rates and intake, and adding water to TMR to achieve a dry matter less than 65% tended to decrease DMI without reducing sorting.

Effect of different forage sources on performance and feeding behavior of Holstein calves.

One hundred seventy-nine Holstein male calves [44.7 kg of body weight (BW) and 8.3 d of age] participated in a series of 3 experiments to evaluate the effect of different forage sources on performance, apparent digestibility, and feeding behavior. Animals in each study were randomly assigned to 1 of 3 different dietary treatments: control (CON) calves were fed starter feed without any forage provision (this treatment was repeated in each of the 3 experiments), and the 2 other treatments consisted of the same starter feed plus a forage source: chopped alfalfa (AH) or rye-grass hay (RH) in the first study; chopped oat hay (OH) or chopped barley straw (BS) in the second study; corn silage (CS) or triticale silage (TS) in the third study. All calves were offered 2L of milk replacer (MR) at 12.5% dry matter (DM) twice daily via a bottle until 50 d of age, and 2L of MR at 12.5% DM during the week before weaning (57 d of age). The study finished when calves were 71 d old. Starter feed, MR, and forage intakes were recorded daily and BW weekly. Calves were individually housed and bedded with wood shavings. Compared with CON, animals receiving OH, TS, and BS consumed more starter feed (0.88 vs. 1.14, 1.17, 1.06 kg/d, respectively) and had greater average daily gain (0.72 vs. 0.93, 0.88, 0.88 kg/d, respectively). Animals in treatments RH, BS, CS, and TS consumed less forage (51 g/d) than AH (120 g/d) and OH (101 g/d) calves. Apparent organic matter, DM, and neutral detergent fiber digestibilities did not differ among treatments (81.5, 81.1, and 54.4%, respectively). Apparent crude protein digestibility was greater in RH, CS, and AH treatments than in CON (80.5 vs. 76.4%, respectively). Compared with CON calves, animals in the AH treatment spent less time eating starter feed and lying, animals in AH and RH treatments spent more time ruminating, with odds ratios (OR) of 5.24 and 5.40, respectively. The AH and RH calves devoted less time to performing nonnutritive oral behaviors (OR: 0.38 and 0.34, respectively), and TS calves tended to devote less time to perform nonnutritive oral behaviors (OR: 0.21) 1h after being offered MR and solid feed. In conclusion, free-choice provision of a forage source to young calves improves feed intake and performance without impairing digestibilities of DM, organic matter, crude protein, and neutral detergent fiber, and, depending on forage source, reduces nonnutritive oral behaviors and stimulates rumination.