Assessment of the safety and immunogenicity of Rhodococcus equi-secreted proteins combined with either a liquid nanoparticle (IMS 3012) or a polymeric (PET GEL A) water-based adjuvant in adult horses and foals--identification of promising new candidate antigens.

Rhodococcus equi is the most common infectious cause of mortality in foals between 1 and 6 months of age. Because of an increase in the number of antibiotic-resistant strains, the optimization of a prophylactic strategy is a key factor in the comprehensive management of R. equi pneumonia. The objectives of this study were to assess the safety and immunogenicity of R. equi-secreted proteins (ReSP) co-administered with either the nanoparticular adjuvant Montanide™ IMS 3012 VG, or a new polymeric adjuvant Montanide™ PET GEL A, and to further investigate the most immunogenic proteins for subsequent immunization/challenge experiments in the development of a vaccine against rhodoccocal pneumonia. The approach involved two phases. The first phase aimed to investigate the safety of vaccination in six adult horses. The second phase aimed to determine the safety and immunogenicity of vaccination in twelve 3-week-old foals. We set out to develop a method based on ultrasound measurements for safety assessment in adult horses in order to evaluate any in situ changes at the injection site, in the skin or the underlying muscle, with quantitative and qualitative data revealing that administration of ReSP combined with the Pet Gel A adjuvant led to an increase in local inflammation, associated with 4- to 7-fold higher levels of anti-R. equi IgGa, IgGb and IgGT, compared to administration of ReSP associated with IMS 3012 adjuvant, but without any impact on animal demeanor. Investigations were then performed in foals with serological and clinical follow-up until 6 months of age. Interestingly, we observed in foals a much lower incidence of adverse local tissue reactions at the injection site than in adult horses, with transient and moderate swelling for the group that received ReSP combined with Pet Gel A. Immunized foals with Pet Gel A adjuvant exhibited a similar response in both IgGa and IgGT levels, but a lower response in IgGb levels, compared to adult horses, with a subisotype profile that may however reflect a bias favorable to R. equi resistance. From the crude extract of secreted proteins, dot-blot screening enabled identification of cholesterol oxidase, mycolyl transferase 3, and PSP (probable secreted protein) as the most immunogenic candidates. Taken together, these results are encouraging in developing a vaccine for foals.

Stochastic modeling in toxicokinetics. Application to the in vivo micronucleus assay.

A stochastic model for the in vivo micronucleus assay is presented. This model describes the kinetic of the rate of micronucleated polychromatic erythrocytes induced by the administration of a mutagenic compound. For this, biological assumptions are made both on the erythropoietic system and on the mechanisms of action of the compound. Its pharmacokinetic profile is also taken into account and it is linked to the induced toxicological effect. This model has been evaluated by analyzing the induction of micronuclei is mice bone marrow by a mutagenic compound, 6-mercaptopurine (6-mp). This analysis enabled to make interesting remarks about the induction of micronuclei by 6-mp and to put to light an unsuspected wavy kinetic by optimizing the experimental design of the in vivo micronucleus assay.

Determination of four nitroimidazole residues in poultry meat by liquid chromatography-mass spectrometry.

A multi-residue liquid chromatography-mass spectrometry (LC-MS) assay method is described for the determination of four nitroimidazoles in poultry muscle. The extraction procedure is based on liquid-liquid extraction with ethyl acetate followed by an evaporation step. A deuterated internal standard is used. The LC separation was made on a C18 bonded silica column with an aqueous formic acid (0.2%) solution-methanol-acetonitrile (81:13:6) mobile phase. Following electrospray ionization, the protonated molecular ion [M+H]+ is obtained for each compound. Monitoring several ions for each nitroimidazole provides the specificity required for confirmatory assay. Validation of the method was performed to estimate linearity, intra-day and inter-day repeatability, accuracy and detection limit. The present method is capable of identifying nitroimidazole residues in muscle at levels below 5 microg/kg.

[Bioavailability of muscle creatine kinase in sheep. Application to the assessment of local tolerance to injectable veterinary formulations]. / Biodisponibilité de la créatine kinase musculaire chez le mouton. Application à l'évaluation de la tolérance locale de formulations vétérinaires injectables.

Pharmacokinetic variables of skeletal muscle creatine kinase were determined in sheep after intravenous and intramuscular administration of the semipurified enzyme. Catheters implanted in the jugular vein were used for both intravenous injections and blood withdrawals. Blood sample collection by vacutainer and hemolysis may in fact have considerable effects on the measurement of creatine kinase activity in plasma. The change in the enzyme activity versus time in the plasma, after intravenous administration (123 +/- 38 U/kg) of creatine kinase, was fitted by a biexponential model. The mean volume of the central compartment (45 +/- 5 mL/kg) was approximately equal to the plasma volume. Plasma half-life and plasma clearance of creatine kinase were 3.7 +/- 1.7 h and 23 +/- 8 mL.kg-1.h-1, respectively. Mean plasma bioavailability of creatine kinase after intramuscular administration (357 +/- 36 U/kg) in both the loins and the gluteal mass was 42%. Maximal plasma activity was observed 4 and 5 h after injection and the half-life of the terminal phase was 7.3 or 8.6 h according to the muscle. The extent of muscle damage after intramuscular administrations of 21 veterinary drug formulations (one product per animal) was estimated from the total creatine kinase activity released in plasma during the 72 h following the injection. Equivalent weights of damaged muscle ranged from 1.4 to 83.3 g according to the irritant potency of the test formulation. Results differed only moderately between the injection sites (right and left gluteal mass) in the same animal. It can be concluded from this study that, in sheep: i) the bioavailability of creatine kinase from different injection sites (gluteal mass and loins) is comparable; and ii) the intra-individual variability in the estimation of muscle damage is moderate. Once validated, this non-invasive approach for local tolerance studies could be of value in assessing and comparing the irritant potency of veterinary drugs and in reducing the number of animals required.