Imaging intracellular fluorescent proteins at nanometer resolution

We introduce a method for optically imaging intracellular proteins at nanometer spatial resolution. Numerous sparse subsets of photoactivatable fluorescent protein molecules were activated, localized (to approximately 2 to 25 nanometers), and then bleached. The aggregate position information from all subsets was then assembled into a superresolution image. We used this method--termed photoactivated localization microscopy--to image specific target proteins in thin sections of lysosomes and mitochondria; in fixed whole cells, we imaged vinculin at focal adhesions, actin within a lamellipodium, and the distribution of the retroviral protein Gag at the plasma membrane.     

Tritrichomonas foetus isolates from cats and cattle show minor genetic differences in unrelated loci ITS-2 and EF-1α

The protozoan parasite Tritrichomonas foetus is well known as an important causative agent of infertility and abortion in cattle (bovine trichomonosis). This World Organisation for Animal Health (O.I.E.) notifiable disease is thought to be under control in many countries including Switzerland. In recent studies, however, T. foetus has also been identified as an intestinal parasite that causes chronic large-bowel diarrhoea in cats. Since the feline isolates were considered indistinguishable from bovine isolates, the possibility and risk of parasite transmission from cats to cattle and vice versa has been intensively discussed in current literature. Therefore, we investigated if cat and cattle isolates are genetically distinct from each other or in fact represent identical genotypes. For this purpose, two independent genetic loci were selected that turned out to be well-suited for a PCR sequencing-based genotyping of trichomonad isolates: (i) previously published internal transcribed spacer region 2 (ITS-2) and (ii) a semi-conserved sequence stretch of the elongation factor-1 alpha (EF-1α) gene used for the first time in the present study. Respective comparative analyses revealed that both loci were sufficiently variable to allow unambiguous genetic discrimination between different trichomonad species. Comparison of both genetic loci confirmed that T. suis and T. mobilensis are phylogenetically very close to T. foetus. Moreover, these two genetic markers were suited to define host-specific genotypes of T. foetus. Both loci showed single base differences between cat and cattle isolates but showed full sequence identity within strains from either cat or cattle isolates. Furthermore, an additional PCR with a forward primer designed to specifically amplify the bovine sequence of EF-1α was able to discriminate bovine isolates of T. foetus from feline isolates and also from other trichomonads. The implications these minor genetic differences may have on the biological properties of the distinct isolates remain to be investigated.     

Body condition score at parturition and postpartum supplemental fat effects on cow and calf performance

Three-year-old Angus x Gelbvieh beef cows nutritionally managed to achieve a BCS of 4 +/- 0.07 (479.3 +/- 36.3 kg of BW) or 6 +/- 0.07 (579.6 +/- 53.1 kg of BW) at parturition were used in a 2-yr experiment (n = 36/yr) to determine the effects of prepartum energy balance and postpartum lipid supplementation on cow and calf performance. Beginning 3 d postpartum, cows within each BCS were assigned randomly to be fed hay and a low-fat control supplement or supplements with either high-linoleate cracked safflower seeds or high-oleate cracked safflower seeds until d 60 of lactation. Diets were formulated to be isonitrogenous and isocaloric, and safflower seed supplements were provided to achieve 5% of DMI as fat. Ultrasonic 12th rib fat and LM area were lower (P < 0.001) for cows in BCS 4 compared with BCS 6 cows throughout the study. Cows in BCS 4 at parturition maintained (P = 0.02) condition over the course of the study, whereas cows in BCS 6 lost condition. No differences (P = 0.44 to 0.71) were detected for milk yield, milk energy, milk fat percentage, or milk lactose percentage because of BCS; however, milk protein percentage was less (P = 0.03) for BCS 4 cows. First-service conception rates did not differ (P = 0.22) because of BCS at parturition, but overall pregnancy rate was greater (P = 0.02) in BCS 6 cows. No differences (P = 0.48 to 0.83) were detected in calf birth weight or ADG because of BCS at parturition. Dietary lipid supplementation did not influence (P = 0.23 to 0.96) cow BW change, BCS change, 12th rib fat, LM area, milk yield, milk energy, milk fat percentage, milk lactose percentage, first service conception, overall pregnancy rates, or calf performance. Although cows in BCS of 4 at parturition seemed capable of maintaining BCS during lactation, the overall decrease in pregnancy rate indicates cows should be managed to achieve a BCS >4 before parturition to improve reproductive success.     

Effect of restricted forage intake on ruminal disappearance of bromegrass hay and a blood meal, feather meal, and fish meal supplement

Two experiments were conducted to determine in situ disappearance of bromegrass hay and a ruminally undegraded protein (RUP) supplement in beef cattle fed restricted amounts of forage. Six Angus crossbred cattle (BW = 589 +/- 44.4 kg; three steers and three heifers) fitted with ruminal cannulas were fed chopped (2.54 cm) bromegrass hay (8.9% CP) at one of three percentages of maintenance intake (30, 55, or 80%; one steer and one heifer per treatment). In both experiments, the cattle were allowed 7 d for diet adaptation followed by 3 d of sample collection. In Exp 1, in situ bags (50 microm pore size) containing 4.1 g of brome-grass hay (OM basis) were inserted into the rumen and subsequently removed at 3, 6, 9, 12, 15, 18, 24, 36, and 48 h after insertion. Nonlinear regression models were used to determine the rapidly solubilized protein Fraction A, the potentially ruminal degradable protein Fraction B, the ruminally undegraded protein Fraction C, and protein degradation rate. Intake level did not affect (P = 0.15 to 0.95) forage protein remaining after in situ incubation or Fractions A, B, and C; however, effective ruminal degradation of hay protein tended to increase quadratically (P = 0.12) as forage intake increased. In Exp 2, 4.2 g (OM basis) of an RUP supplement (6.8% porcine blood meal, 24.5% hydrolyzed feather meal, and 68.7% menhaden fish meal) formulated to provide equal amounts of metabolizable protein across all levels of hay consumption was evaluated in a similar manner as in Exp 1. The undegraded protein fraction of the supplement did not differ (P = 0.16 to 0.74) across treatments at 3, 6, 9, and 18 h; however, increasing forage intake resulted in a linear increase (P < or = 0.06) in undegraded protein remaining at 12, 15, 24, 36, and 48 h. Dietary treatment had no affect (P = 0.30) on protein Fractions A, B, or C; however, protein degradation rate of the supplement decreased linearly (P = 0.03) as forage intake increased. Therefore, effective ruminal degradation of the supplement decreased linearly (P = 0.01) from 50.8 to 40.9% as forage intake increased from 30 to 80% of maintenance. Corresponding estimates of supplement RUP were 49.2, 56.5, and 59.1% for the 30, 55, and 80% of maintenance intake treatments, respectively. Restricting dietary intake can decrease the quantity of dietary protein that escapes ruminal degradation. Tabular estimates of RUP may not be appropriate for formulating diets to balance metabolizable protein in beef cattle consuming limited quantities of forage.