Dose determination and confirmation of a long-acting formulation of ceftiofur (ceftiofur crystalline free acid) administered subcutaneously for the treatment of bovine respiratory disease.

The objective of this work was to determine and confirm an effective dose of ceftiofur crystalline free acid sterile oil suspension (CCFA-SS, 100 mg ceftiofur equivalents (CE)/mL], a long-acting single-administration ceftiofur formulation, for the treatment of the bacterial component of bovine respiratory disease (BRD). Study 1 was a dose determination study that used an intratracheal Mannheimia haemolytica (Pasteurella haemolytica) challenge model to evaluate single-administration doses of CCFA-SS at 0.0, 1.1, 2.2, 3.3, 4.4 or 5.5 mg CE/kg body weight (BW) for the treatment of BRD. Data from this study were used to select doses for field testing in three multi-location clinical studies. In Study 2, the efficacy of a single administration dose of CCFA-SS at 4.4 mg CE/kg BW was compared with a negative control for the treatment of naturally occurring BRD in feedlot cattle. Treatments were administered when uniform clinical signs of BRD were present. Study 3 used a design similar to Study 2, and compared single-administration doses of CCFA-SS at 3.0 or 4.4 mg CE/kg BW with the positive-control tilmicosin (Micotil(R) 300 Injection, Elanco Animal Health) at 10 mg/kg BW. Study 4 compared the efficacy of single doses of CCFA-SS of 1.1-8.8 mg CE/kg BW with tilmicosin at 10 mg/kg BW. A total of 1176 cattle were included in these clinical studies. In Study 1, a dose of 4.55 mg CE/kg BW was determined to be effective. This was rounded to 4.4 mg CE/kg for field testing. In Study 2, a single dose of CCFA-SS at 4.4 mg CE/kg BW had a higher treatment success rate on day 14 (61%) than negative controls (26%, P < 0.01). However, in Study 3 this dose was judged to be at the beginning of an efficacious dose range for the treatment of BRD when compared with tilmicosin. In Study 4, day 28 treatment success rates were higher for CCFA-SS at 4.4-8.8 CE/kg BW than for tilmicosin (P=0.002) or the noneffective CCFA-SS dose of 1.1 mg CE/kg BW (P < 0.001). Based on decision criteria for Study 4, the effective dose was determined to be 4.4-5.5 mg CE/kg BW. These clinical studies demonstrated that a single dose of CCFA-SS (100 mg CE/mL) administered subcutaneously (s.c.) in the neck at 4.4-5.5 mg CE/kg BW is an effective treatment for BRD in feedlot cattle. However, this route of administration is no longer being considered for this formulation because of the ceftiofur residues that are present at the injection site for extended periods of time.

Identification of Actinobacillus pleuropneumoniae virulence genes using signature-tagged mutagenesis in a swine infection model.

Actinobacillus pleuropneumoniae is a significant respiratory pathogen of swine causing a severe and often fatal fibrinous hemorrhagic bronchopneumonia with significant economic losses resulting from chronic as well as acute infections. This study describes the application of a signature-tagged mutagenesis (STM) system to identify in vivo critical genes of A. pleuropneumoniae. Twenty pools representing over 800 A. pleuropneumoniae mutants were screened in a natural-host porcine infection model and presumptive attenuated mutants were selected. The identity of the disrupted gene in each mutant was determined using an inverse PCR approach to amplify DNA sequences adjacent to the transposon insertion, followed by sequencing of the PCR product and comparison to bacterial databases. In vitro and in vivo competitive indices were determined for each unique mutant, and a total of 20 unique, attenuating gene disruptions were identified including insertions into homologues of genes involved in biosynthesis, virulence determinants, regulation, translation and unknown functions. Three of the genes required for virulence of A. pleuropneumoniae in this study were also identified in a previous STM study of Pasteurella multocida. Seven of the STM-derived mutants were also evaluated for their potential as live vaccine strains and provided good protection against homologous challenge.

Position 2 and position 2/Ala15-substituted analogs of bovine growth hormone-releasing factor (bGRF) with enhanced metabolic stability and improved in vivo bioactivity.

In order to prepare GRF analogs with high activity in vivo, a strategy was undertaken to stabilize the peptide to dipeptidylpeptidase IV (DPP-IV), a protease found in plasma which inactivates native human and bovine GRF by cleavage of the Ala2-Asp3 bond. Replacement of the Ala2 residue with Ser, Thr, or Gly in [Leu27]bGRF(1-29)NH2 resulted in peptides greatly stabilized against proteolysis in plasma, but having low inherent GH-releasing activity when tested in bovine pituitary cell cultures. Replacement of Gly15 with Ala15 was marginally effective in improving the in vitro bioactivity of this group of peptides. When tested for GH-hormone release in steers, however, the Thr2,Ala15 analog was four times more potent than bGRF(1-44)NH2. Eleven additional analogs from the [X2,Ala15,Leu27]bGRF(1-29)NH2 series were synthesized and evaluated for metabolic stability in bovine plasma and for GH releasing activity in steers in vivo and in rat pituitary cells in vitro. Two compounds, [Val2,Ala15,Leu27]dGRF(1-29)NH2 and [Ile2,Ala15,Leu27]-bGRF(1-29)NH2, had increased GH-releasing activity in steers over that of [Thr2,Ala15,Leu27]-bGRF(1-29)NH2 and over a previously reported super-potent analog, [desNH2Tyr1,D-Ala2,Ala15]-hGRF(1-29)NH2.

Growth hormone-releasing factor analogs with hydrophobic residues at position 19. Effects on growth hormone releasing activity in vitro and in vivo, stability in blood plasma in vitro, and secondary structure.

To test the hypothesis that replacing Ala19 in growth hormone-releasing factor (GRF) with more hydrophobic residues will increase growth hormone releasing activity, four GRF analogs were prepared and tested. The molecules were made by substituting Val, Ile, or Leu at position 19 of [Thr2,Ala15,Leu27]bGRF(1-29)NH2. The compounds were evaluated for growth hormone (GH) releasing activity in vitro (rat anterior pituitary cells) and in vivo (steers). Additionally, their half-life in vitro was determined in bovine plasma, and their secondary structure was examined by circular dichroism. In pituitary cells, peptides with substitutions at position 19 had the following potencies: Ala (native), 0.37; Val, 1.16; Ile, 0.37; Leu, 0.043. When assayed in steers as a single iv bolus, over a 2-h period, the compounds gave the following integrated GH response: Ala, 2.75; Val, 2.67; Ile, 2.57; Leu, 1.55. Only the Leu analog was statistically different from the other three (p = 0.05). In bovine plasma, the half-lives (hours) were as follows: Ala, 4.9; Val, 6.6; Ile, 12.3; Leu, 14.7. In phosphate buffer the compounds were calculated to have the following percent helical content: Ala, 26; Val, 21; Ile, 27; Leu, 32. For these analogs, helicity in aqueous buffer is inversely related to their in vitro activity. Using a linear multiple regression model, the plasma half-life of the analogs positively correlated (r2 = 0.999) with both the hydrophobicity of the residue at position 19 and the helicity of the analog. Although the Val analog had both increased inherent activity and increased plasma stability in vitro compared to the Ala analog, in this study we were unable to demonstrate an increase in activity in vivo. The in vivo GH releasing activity of the analogs was not simply related to a combination of their intrinsic GH releasing activity and their in vitro plasma half-life. This suggests that in vivo additional factors are moderating the expression of activity.

Recombinant bovine somatotropin improves growth performance in finishing beef steers.

The objectives of this study were to determine the efficacy of various doses of rbST on ADG and feed efficiency (FE) and to describe carcass composition changes in finishing beef steers. In Exp. 1, 96 crossbred beef steers (393 kg) received daily i.m. injections of buffer or 33, 100, or 300 micrograms/kg of BW of rbST (0ST, 33ST, 100ST, 300ST). In Exp. 2, 200 crossbred beef steers (417 kg) received daily i.m. injections of buffer or 8.25, 16.5, 33, or 66 micrograms/kg of BW or rbST (0ST, 8.25ST, 16.5ST, 33ST, 66ST). Treatments were administered until steer BW per pen averaged 540 kg in Exp. 1 and 560 kg in Exp. 2. An 86% concentrate: 14% roughage diet was fed once daily (CP: 16.5% in Exp. 1, 20.2% in Exp. 2). In Exp. 1, growth performance of steers receiving rbST was dose-dependent; ADG changed linearly (P = .01), DMI decreased linearly (P = .03), and FE changed quadratically (P less than .03). The 33ST steers responded with improved ADG and FE, 100ST with improved FE, and 300ST with lower ADG and poorer FE, compared with 0ST. In Exp. 2, the ADG response was quadratic (P = .01), DMI decreased linearly (P = .003), and FE improved quadratically (P = .004) with increasing dose of rbST. Steers receiving 16.5ST and 33ST responded with improved ADG and FE, whereas steers receiving 8.25ST and 66ST responded with improved FE but not ADG relative to 0ST steers. In Exp. 1 and 2, rbST administration altered carcass composition by increasing carcass protein and decreasing carcass fat.(ABSTRACT TRUNCATED AT 250 WORDS)

Food intake alters the serum growth hormone response to bovine growth hormone-releasing factor in meal-fed Holstein steers.

The hypothesis that endocrine or nutritional factors related to feeding may affect pituitary responsiveness to an acute challenge with bovine GH-releasing factor (1-44)-NH2 (GRF) was examined in steers. In these experiments, either steers were trained to consume their total daily food allotment in a 2-h period (meal-fed) or food was withheld at the normal time of feeding (sham-fed). In the first of three experiments, the serum GH pattern was determined around the time of feeding in meal-fed and sham-fed steers. The temporal GH rhythm in both groups appeared to be synchronized to the time of feeding, with limited pulsatile GH activity occurring 2-3 h after feeding. Baseline secretion of GH and total area under the GH response curve were lower (P less than 0.01) in meal-fed compared with sham-fed steers. In the second experiment, 50 micrograms GRF was injected i.v. in meal-fed steers at -4, -2, 0, +2, +4, +6 and +8 h relative to the time of feeding. The number of steers responding to GRF (53%), the amplitude of the GH peak (15.8 micrograms/l) and the area under the GH response curve (0.6 arbitrary units) were lower (P less than 0.001) after than before feeding (90 +/- 6 (S.E.M.)%, 61.3 +/- 3.2 micrograms/l and 2.0 +/- 0.3 units respectively). Of those steers responding to GRF, the GH response was significantly reduced following feeding compared with before feeding. In the third experiment, 50 micrograms GRF was injected i.v. in sham-fed steers at -4, -2, 0, +4 and +6 h relative to the time of sham-feeding.(ABSTRACT TRUNCATED AT 250 WORDS)