Conduct,Oversight, and Ethical Considerations of Clinical Trials in Companion Animals with Cancer: Report of a Workshop on Best Practice Recommendations

Development of effective and safe treatments for companion animals with cancer requires the collaboration of numerous animal health professionals and the full engagement of animal owners. Establishing ‘Best Practice Recommendations’ for clinical trials in veterinary oncology represents an important step toward meeting the goal of rigorous clinical trial design and conduct that is required to establish valid evidence. Likewise, optimizing patient welfare and owner education and advocacy is crucial to meet the unique ethical obligations to both owners and animals enrolled in these clinical trials and to ensure trust in the team conducting the research. To date, ‘Best Practice Recommendations’ for clinical trial conduct have not been reported for veterinary oncology. This document summarizes the consensus of a workshop held in November, 2014 to identify relevant ethical principles and to ensure responsible conduct of clinical research in companion animals with cancer. It is intended as a working document that will be updated as advances in science and ethical considerations require. To the extent possible, existing guidelines for the conduct and oversight of clinical trials in humans have been adapted for veterinary trials to avoid duplicative effort and to facilitate integration of clinical trials such that translational research with benefits for both companion animals and humans are encouraged.     

A Quantitative Polymerase Chain Reaction Assay for the Detection and Quantification of Epizootic Epitheliotropic Disease Virus (EEDV; Salmonid Herpesvirus 3)

Epizootic epitheliotropic disease virus (EEDV; salmonid herpesvirus [SalHV3]; family Alloherpesviridae) causes a systemic disease of juvenile and yearling Lake Trout Salvelinus namaycush. No cell lines are currently available for the culture and propagation of EEDV, so primary diagnosis is limited to PCR and electron microscopy. To better understand the pervasiveness of EEDV (carrier or latent state of infection) in domesticated and wild Lake Trout populations, we developed a sensitive TaqMan quantitative PCR (qPCR) assay to detect the presence of the EEDV terminase gene in Lake Trout tissues. This assay was able to detect a linear standard curve over nine logs of plasmid dilution and was sensitive enough to detect single-digit copies of EEDV. The efficiency of the PCR assay was 99.4 ± 0.06% (mean ± SD), with a 95% confidence limit of 0.0296 (R² = 0.994). Methods were successfully applied to collect preliminary data from a number of species and water bodies in the states of Pennsylvania, New York, and Vermont, indicating that EEDV is more common in wild fish than previously known. In addition, through the development of this qPCR assay, we detected EEDV in a new salmonid species, the Cisco Coregonus artedi. The qPCR assay was unexpectedly able to detect two additional herpesviruses, the Atlantic Salmon papillomatosis virus (ASPV; SalHV4) and the Namaycush herpesvirus (NamHV; SalHV5), which both share high sequence identity with the EEDV terminase gene. With these unexpected findings, we subsequently designed three primer sets to confirm initial TaqMan qPCR assay positives and to differentiate among EEDV, ASPV, and NamHV by detecting the glycoprotein genes via SYBR Green qPCR.

Received April 20, 2015; accepted November 10, 2015     

Impact of variation in structure of condensed tannins from sainfoin (Onobrychis viciifolia) on in vitro ruminal methane production and fermentation characteristics

Our study investigated the effects of condensed tannins (CT) on rumen in vitro methane (CH₄) production and fermentation characteristics by incubating lucerne in buffered rumen fluid in combination with different CT extracts at 0 (control), 40, 80 and 120 g CT/kg of substrate DM. Condensed tannins were extracted from four sainfoin accessions: Rees ‘A’, CPI63763, Cotswold Common and CPI63767. Gas production (GP) was measured using a fully automated GP apparatus with CH₄ measured at distinct time points. Condensed tannins differed substantially in terms of polymer size and varied from 13 (Rees ‘A’) to 73 (CPI63767) mean degree of polymerization, but had relatively similar characteristics in terms of CT content, procyanidin: prodelphinidin (PC: PD) and cis:trans ratios. Compared to control, addition of CT from CPI63767 and CPI63763 at 80 and 120 g CT/kg of substrate DM reduced CH₄ by 43% and 65%, and by 23% and 57%, respectively, after 24‐h incubation. Similarly, CT from Rees ‘A’ and Cotswold Common reduced CH₄ by 26% and 46%, and by 28% and 46% respectively. Addition of increasing level of CT linearly reduced the maximum rates of GP and CH₄ production, and the estimated in vitro organic matter digestibility. There was a negative linear and quadratic (p < 0.01) relation between CT concentration and total volatile fatty acid (VFA) production. Inclusion of 80 and 120 g CT/kg of substrate DM reduced (p < 0.001) branched‐chain VFA production and acetate: propionate ratio and was lowest for CPI63767. A decrease in proteolytic activity as indirectly shown by a change in VFA composition favouring a shift towards propionate and reduction in branched‐chain VFA production varied with type of CT and was highest for CPI63767. In conclusion, these results suggest that tannin polymer size is an important factor affecting in vitro CH₄ production which may be linked to the CT interaction with dietary substrate or microbial cells.     

Deoxynivalenol in chicken feed alters the vaccinal immune response and clinical biochemical serum parameters but not the intestinal and carcass characteristics

This study was conducted to investigate the impacts of deoxynivalenol (DON) feeding either alone or in combination with a microbial feed additive (MFA) on the immune response to a viral vaccine and serum clinical chemical parameters. Forty 1‐day‐old boiler chicks were weighed and randomly divided into four groups, 10 birds in each group: (i) control group fed with basal diet; (ii) DON group fed with basal diet artificially contaminated with 10 mg DON/kg feed; (iii) DON + MFA group fed with basal diet contaminated with 10 mg DON/kg feed and supplemented with 2.5 kg of MFA/ton feed; and (iv) MFA group fed with basal diet supplemented with 2.5 kg of MFA/ton feed. At 35 days of age, birds were slaughtered and blood was collected for investigating the antibody titre against infectious bronchitis virus (IBV) and clinical chemical parameters. The results showed that DON reduced (p = 0.032) the titre against IBV, decreased (p = 0.005) the level of alanine transaminase (ALT) (4.2 ± 0.5 U/l) compared with control birds (6.4 ± 0.5 U/l), increased (p = 0.002) the serum cholesterol concentration (144 ± 6 mg/dl) compared with their control counterparts (123 ± 5 mg/dl) and increased (p = 0.074) the amount of circulating triglycerides (62.25 ± 7.50 mg/dl) compared with controls (39.55 ± 4.74). These results indicate that dietary DON altered the humoral immune response to viral vaccine and affected the serum clinical biochemistry. However, DON in combination with MFA did not affect serum IBV titre. Taken together, DON in the feed of broilers produced an impairment of the success of IBV vaccine and affected the health of birds.     

The influences of ambient temperature and crude protein levels on performance and serum biochemical parameters in broilers

This study was undertaken to investigate the effects of ambient temperature, crude protein levels and their interaction on performance and serum biochemical parameters of broiler chickens. A total of 216 Arbor Acre broiler chickens (108 males and 108 females) were used in a 2 × 3 factorial arrangement and randomly reared at two temperatures (normal temperature: 23 °C; daily cyclic high temperature: 28–32 °C) and fed on three diets with different crude protein levels (153.3, 183.3 or 213.3 g/kg, with constant essential amino acids) from 28 to 42 days of age. Daily cyclic high ambient temperature decreased final body weight, average daily weight gain, average daily feed intake and serum total protein contents (p < 0.001, p < 0.001, p < 0.001, p = 0.008 respectively), but increased feed/gain, mortality, respiratory rate, rectal temperature, serum uric acid contents and serum creatine kinase activity (p = 0.008, p = 0.003, p < 0.0001, p < 0.0001, p < 0.0001, p = 0.003 respectively), irrespective of crude protein levels. At the ambient temperature, reducing crude protein levels resulted in an increase in feed/gain (p < 0.001), but a decrease in serum total protein and uric acid contents. Only serum creatine kinase activity in broiler chickens was interacted by daily cyclic high ambient temperature and dietary crude protein levels (p = 0.003). These results indicated that daily cyclic high ambient temperature had a great effect on performance and serum biochemical parameters in broiler chickens, whereas dietary crude protein levels affected them partially.     

Mineral requirements of hair sheep in tropical climates

In this study, we evaluated the requirements of calcium (Ca), phosphorus (P), magnesium (Mg), sodium (Na) and potassium (K) for sheep hair growth. Experimental diets contained different levels of metabolizable energy [ME; 0.96, 1.28, 1.72, 2.18 and 2.62 Mcal/kg of dry matter, (DM), corresponding to 4.23, 5.64, 7.58, 9.61 and 11.55 MJ/kg DM]. The lambs' hair (n = 48) at 2 months of age presented an average body weight (BW) of 12.05 ± 1.81 kg. At the beginning of the experiment, eight animals were slaughtered as a reference group to estimate the initial empty body weight and body composition. Net mineral requirements (g/day) ranged from 0.73 to 0.71 g of Ca, 0.51 to 0.49 g of P, 0.026 to 0.026 g of Mg, 0.16 to 0.19 g of Na and 0.15 to 0.13 g of K for animals with a BW ranging from 15 to 30 kg and a daily gain of 100 g. The results of this study indicate that the net macromineral requirements for weight gain in Morada Nova lambs are different from the values commonly recommended by the Agricultural and Food Research Council.     

Two single nucleotide polymorphisms in the promoter of the ovine myostatin gene (MSTN) and their effect on growth and carcass muscle traits in New Zealand Romney sheep

Myostatin is a negative regulator of muscle growth and development in mammals, and variation in ovine myostatin gene (MSTN) has been demonstrated to be associated with variation in the muscularity of sheep. Polymerase chain reaction–single‐stranded conformational polymorphism (PCR–SSCP) was used to look for single nucleotide polymorphisms (SNPs) in a 304‐bp amplicon from the promoter region of ovine MSTN. Sequence analyses revealed two previously identified SNPs (c.?2449G/C and c. ?2379T/C) that resulted in three haplotypes (H₁ (c.[?2449G; ?2379C]), H₂ (c.[?2449C; ?2379C]) and H₃ (c.[?2449G; ?2379T]). The effect of these SNPs on growth and carcass traits was investigated in 357 NZ Romney lambs. General linear mixed‐effect models revealed that sheep with the genotype c.?2449GC had a higher loin meat yield (p = 0.032) and proportion loin yield (p = 0.028), than those with the genotype c.?2449GG. The genotype c.?2379CC was associated with an increase in three weight traits: birthweight (p = 0.003), tailing weight (p = 0.009) and weaning weight (p = 0.028), when compared with the genotype c.?2379TC, but it was not found to have an association with growth rate. This suggests that c.?2379T/C has an effect that originates at, or before birth. Haplotype H₃ was associated with a decrease in birthweight (p = 0.002), tailing weight (p = 0.003) and weaning weight (p = 0.011). Haplotype H₂ was associated with increased loin yield (p = 0.012) and proportion loin yield (p = 0.002). The SNPs may have value as genetic markers for improved Romney breeding.