Ionophore strategy affects growth performance and carcass characteristics in feedlot steers.

One hundred ninety-two steers (BW = 354 ± 23.5 kg) were used in a randomized block design to evaluate the effects of ionophore and ractopamine hydrochloride (RH) supplementation strategies on performance and carcass characteristics. Twelve pens of 4 steers were assigned to each of the following treatments: unsupplemented control (CON), laidlomycin propionate (12.1 mg/kg DM) with or without RH (LPRH and LP, respectively), and monensin sodium (36.4 mg/kg DM) with RH (MSRH). Steers were fed for 151 d, of which respective treatments received RH (Actogain; Zoetis, Florham Park, NJ) at a rate of 300 mg/(animal · d) for the final 32 d. Laidlomycin was removed from the LPRH treatment during this period, as no combination feeding has been approved. Upon harvest, carcass data were collected by trained personnel, and subsequent analysis of the LM was conducted to estimate tenderness using Warner-Bratzler shear force (WBSF). Prior to RH supplementation, both LP and LPRH had greater ADG ( ≤ 0.02) and G:F ( < 0.01) than CON, whereas MSRH was intermediate. During the final 32 d, MSRH improved G:F ( ≤ 0.02) compared to all other treatments and tended to increase ADG over unsupplemented controls ( = 0.05). Cattle receiving LP without RH had significantly greater BW at d 151 than CON ( = 0.02), whereas both RH treatments tended to improve final BW ( ≤ 0.09). Ionophores improved ADG ( ≤ 0.03) and G:F ( < 0.01) for the entire feeding period, and although LP-supplemented cattle had greater DMI for the final 32 d than both RH treatments ( ≤ 0.01), intakes for the 151-d trial were similar among treatments. Carcass weights were greater ( = 0.04) in cattle fed LP with no RH than CON, where cattle yielded an average of 12 kg more HCW. Ractopamine increased LM area in MSRH-supplemented cattle ( = 0.03) and tended to increase LM area for steers receiving LPRH ( = 0.07). Longissimus steaks of MSRH-supplemented cattle had greater WBSF values than CON ( = 0.04) after 7 d of postmortem aging and greater WBSF values than LPRH steaks after 28 d ( = 0.03). All other carcass and WBSF measurements were similar among treatments. The results of this study indicate that LP supplementation without RH may yield a performance similar to and carcass responses associated with the administration of a ß-agonist. These results also suggest that performance and carcass characteristics for cattle fed LP are similar to those of cattle fed monensin throughout the feeding period.

Comparison of tulathromycin and tilmicosin on the prevalence and severity of bovine respiratory disease in feedlot cattle in association with feedlot performance, carcass characteristics, and economic factors.

The objectives of this study were to 1) quantify effects of metaphylactic treatment for bovine respiratory disease (BRD) on growth performance, carcass characteristics, and lung lesion prevalence and severity; 2) evaluate the association of lung lesion prevalence and severity with carcass characteristics; and 3) evaluate effects of therapeutic treatment on carcass characteristics and lung lesion prevalence and severity. The study was conducted at a commercial feedlot in the Texas Panhandle in which steers (n = 2,336) initially weighing 312.1 ± 9.6 kg were sourced from auction markets and allocated in a randomized complete block design to 1 of 3 treatments (no metaphylactic [no antimicrobial drug {ND}] treatment, tilmicosin at 10 mg/kg BW [TIL], and tulathromycin at 2.5 mg/kg BW [TUL]). Lungs of all steers were evaluated during harvest to assess presence and severity of pneumonic lesions in the anteroventral lobes and the presence and severity of pleural adherences. Compared to the ND treatment, steers treated via metaphylactic therapy had greater (P < 0.05) metaphylactic cost, ADG, shrunk final BW, dressed carcass yield, HCW, 12th rib fat, calculated empty body fat (EBF), and gross revenue, concurrent with reduced (P < 0.05) BRD treatment costs and financial losses from BRD death and railed cattle, cumulatively resulting in greater financial returns. Lung lesions were present in 64.3% of lungs and were distributed similarly between metaphylactic treatments (63.9%) and ND (65.1%) cattle. Steers with advanced lung lesions present at harvest were associated with reduced (P < 0.05) HCW, KPH, 12th rib fat, calculated yield grades, marbling scores, and calculated EBF as compared to steers without lung lesions. Steers pulled for BRD had increased (P < 0.01) incidence of advanced lung lesions, mortality, and railers with decreased (P < 0.05) HCW, 12th rib fat, KPH, marbling score, calculated EBF, and percentage choice carcasses when compared to non-BRD event steers. From the results of this study, controlling BRD through the use of metaphylactic treatments on arrival in heavier cattle improved financial returns primarily driven by reductions in cost of death loss and railers.

Adaptation of the WHO guideline for residual DNA in parenteral vaccines produced on continuous cell lines to a limit for oral vaccines.

Although there is a WHO guidance for a limit on residual DNA for parenterally administered vaccines produced on continuous cell lines, there is no corresponding guidance for oral vaccines. To help determine an oral limit, we performed a study of Vero cell DNA uptake in rats, in which the relative uptake and persistence of Vero cell DNA administered orally was compared to its uptake when delivered intramuscularly (IM). The results of this study allowed the generation of an empirically derived IM versus oral factor (10(6)) representing the relative inefficiency of DNA uptake by oral administration. This factor was then applied to the WHO recommended parenteral limit of 10 ng/dose to determine a corresponding upper limit on the level of residual Vero cell DNA for an oral vaccine of 10 mg. As a conservative approach, this empirically determined limit was reduced 100-fold to 100 microg. Thus, the results of this animal study, together with additional evidence in the literature, support a residual DNA safety limit of 100 microg per dose for an oral vaccine produced on a continuous cell line.

Tumorigenicity assessments of Per.C6 cells and of an Ad5-vectored HIV-1 vaccine produced on this continuous cell line.

PER.C6, a cell line derived from human embryonic retinal cells transformed with the Adenovirus Type 5 (Ad5) E1A and E1B genes, is used to produce E1-deleted Ad5 vectors such as the MRKAd5 HIV-1 gag vaccine. While whole, live PER.C6 cells are capable of growing as tumours when transplanted subcutaneously into immunodeficient nude mice at a high dosage, the process for vaccine production includes filtration steps and other methods which effectively preclude contamination by intact viable substrate cells. However, because of the neoplastic nature of this cell line, we carried out a series of investigations to assess the tumorigenic risk posed by residuals from the cell substrate in a vaccine. To address concerns about transmission of oncogenic DNA, we demonstrated that purified PER.C6 cellular DNA does not induce tumours in newborn hamsters or nude mice. To address concerns about other potential residuals, including hypothetical adventitious tumour viruses, we demonstrated that a PER.C6 cell lysate and a MRKAd5 HIV-1 gag vaccine produced on PER.C6 cells do not induce tumours in newborn hamsters or newborn rats. These results, in conjunction with the wide panel of viral safety tests performed on these cells, support the safety of the PER.C6 as a cell substrate for vaccine production.

Detection of integration of plasmid DNA into host genomic DNA following intramuscular injection and electroporation.

Plasmid vectors have been widely used for DNA vaccines and gene therapy. Following intramuscular injection, the plasmid that persists is extrachromosomal and integration into host DNA, if it occurs at all, is negligible. However, new technologies for improving DNA delivery could increase the frequency of integration. In the present study, we tested the effect of electroporation on plasmid uptake and potential integration following intramuscular injection in mice, using a plasmid containing the mouse erythropoietin gene. Electroporation increased plasmid tissue levels by approximately six- to 34-fold. Using a quantitative gel-purification assay for integration, electroporation was found to markedly increase the level of plasmid associated with high-molecular-weight genomic DNA. To confirm integration and identify the insertion sites, we developed a new assay - referred to as repeat-anchored integration capture (RAIC) PCR - that is capable of detecting rare integration events in a complex mixture in vivo. Using this assay, we identified four independent integration events. Sequencing of the insertion sites suggested a random integration process, but with short segments of homology between the vector breakpoint and the insertion site in three of the four cases. This is the first definitive demonstration of integration of plasmid DNA into genomic DNA following injection in vivo.

Plasmid DNA vaccines: investigation of integration into host cellular DNA following intramuscular injection in mice.

The primary safety concern for DNA vaccines is their potential to integrate into the host cell genome. We describe an integration assay based on purification of high-molecular-weight genomic DNA away from free plasmid using gel electrophoresis, such that the genomic DNA can then be assayed for integrated plasmid using a sensitive PCR method. The assay sensitivity was approximately 1 plasmid copy/microg DNA (representing approximately 150,000 diploid cells). Using this assay, we carried out integration studies of three different plasmid DNA vaccines, containing either the influenza hemagglutinin, influenza matrix or HIV gag gene. Six weeks after intramuscular injection, free plasmid was detected in treated muscle at levels ranging from approximately 1,000 to 4,000 copies/microg DNA. At 6 months, the plasmid levels ranged between 200 and 800 copies/microg DNA. Gel purification of genomic DNA revealed that essentially all of the detectable plasmid in treated quadriceps was extrachromosomal. If integration had occurred, the frequency was

Plasmid DNA vaccines: tissue distribution and effects of DNA sequence, adjuvants and delivery method on integration into host DNA.

A variety of factors could affect the frequency of integration of plasmid DNA vaccines into host cellular DNA, including DNA sequences within the plasmid, the expressed gene product (antigen), the formulation, delivery method, route of administration, and the type of cells exposed to the plasmid. In this report, we examined the tissue distribution and potential integration of plasmid DNA vaccines following intramuscular administration in mice and guinea pigs. We compared needle versus Biojector (needleless jet) delivery, examined the effect of aluminum phosphate adjuvants, compared the results of different plasmid DNA vaccines, and tested a gene (the human papilloma virus E7 gene) whose protein product is known to increase integration frequency in vitro. Six weeks following intramuscular injection, the vast majority of the plasmid was detected in the muscle and skin near the injection site; lower levels of plasmid were also detected in the draining lymph nodes. At early time points (1-7 days) after injection, a low level of systemic exposure could be detected. Occasionally, plasmid was detected in gonads, but it dissipated rapidly and was extrachromosomal - indicating a low risk of germline transmission. Aluminum phosphate adjuvant had no effect on the tissue distribution and did not result in a detectable increase in integration frequency. Biojector delivery, compared with needle injection, greatly increased the uptake of plasmid (particularly in skin at the injection site), but did not result in a detectable increase in integration frequency. Finally, injection of a plasmid DNA vaccine containing the human papilloma virus type 16 E7 gene, known to increase integration in vitro, did not result in detectable integration in mice. These results suggest that the risk of integration following intramuscular injection of plasmid DNA is low under a variety of experimental conditions.

Plasmid DNA vaccines: assay for integration into host genomic DNA.

The primary safety concern for DNA vaccines is their potential to integrate into host cellular DNA. We describe a sensitive and quantitative assay for investigating the tissue distribution and integration of plasmid DNA vaccines. By including gonadal tissues in the analysis, the potential for germline transmission is also assessed. At various time points after injection, total DNA is isolated from a variety of tissues and assayed by PCR for the presence of plasmid. To test for integration, genomic DNA is first purified away from free plasmid using a series of different gel electrophoresis procedures. The gel-purified genomic DNA is then assayed for integrated plasmid using PCR. Stringent methods are used to prevent contamination. The assay, validated using a variety of positive and negative controls, is capable of detecting one copy of plasmid per ug DNA (approximately 150,000 diploid cells). Using this assay, we have carried out intramuscular studies in mice or guinea pigs for four different DNA vaccine plasmids. There was no evidence of integration to a sensitivity of about one copy/microg DNA, which is at least three orders of magnitude below the spontaneous mutation frequency.