Six nonlactating and six lactating adult female goats received a single subcutaneous injection of ceftiofur crystalline free acid (CCFA) at a dosage of 6.6 mg/kg. Blood samples were collected from the jugular vein before and at multiple time points after CCFA administration. Milk samples were collected twice daily. Concentrations of ceftiofur and desfuroylceftiofur-related metabolites were measured using high-performance liquid chromatography. Data were analyzed using compartmental and noncompartmental approaches. The pharmacokinetics of CCFA in the domestic goat was best described by a one compartment model. Mean (±SD) pharmacokinetic parameters were as follows for the nonlactating goats: area under the concentration time curve(0-∞) (159 h·μg/mL ± 19), maximum observed serum concentration (2.3 μg/mL ± 1.1), time of maximal observed serum concentration (26.7 h ± 16.5) and terminal elimination half life (36.9 h; harmonic). For the lactating goats, the pharmacokinetic parameters were as follows: area under the concentration time curve(0-∞) (156 h·μg/mL ± 14), maximum observed serum concentration (1.5 μg/mL ± 0.4), time of maximal observed serum concentration (46 h ± 15.9) and terminal elimination half life (37.3 h; harmonic). Ceftiofur and desfuroylceftiofur-related metabolites were only detectable in one milk sample at 36 h following treatment. There were no significant differences in the pharmacokinetic parameter between the nonlactating and lactating goats.

Six adult male alpacas received one subcutaneous administration of ceftiofur crystalline free acid (CCFA) at a dosage of 6.6 mg/kg. After a washout period, the same alpacas received three subcutaneous doses of 6.6 mg/kg CCFA at 5-day intervals. Blood samples collected from the jugular vein before and at multiple time points after each CCFA administration were assayed for ceftiofur- and desfuroylceftiofur-related metabolite concentrations using high-performance liquid chromatography. Pharmacokinetic disposition of CCFA was analyzed by a noncompartmental approach. Mean pharmacokinetic parameters (± SD) following single-dose administration of CCFA were Cmax (2.7 ± 0.9 μg/mL); Tmax (36 ± 0 h); area under the curve AUC0→∞ (199.2 ± 42.1 μg·h/mL); terminal phase rate constant λz (0.02 ± 0.003/h); and terminal phase rate constant half-life t1/2λz (44.7 h; harmonic). Mean terminal pharmacokinetic parameters (±SD) following three administrations of CCFA were Cmax (2.0 ± 0.4 μg/mL); Tmax (17.3 ± 16.3 h); AUC0→∞ (216.8 ± 84.5 μg·h/mL); λz (0.01 ± 0.003/h); and t1/2λz (65.9 h; harmonic). The terminal phase rate constant and the Tmax were significantly different between single and multiple administrations. Local reactions were noted in two alpacas following multiple CCFA administrations.

Despite tremendous progress in our understanding of human immunodeficiency virus (HIV) natural history and advances in HIV treatment, there is neither an approved vaccine nor a cure for infection. Here, we describe the development and characterization of a novel replicating vaccine vector utilizing Cytomegalovirus (CMV) and a TLR5 adjuvant. After partial truncation of the central, immunodominant hypervariable domain, flagellin (fliC) from Salmonella was cloned downstream of a codon optimized gag gene from simian immunodeficiency virus (SIV) and transiently expressed in telomerized rhesus fibroblast (TeloRF) cells in culture. Lysates generated from these transfected cells induced the tumor necrosis factor alpha (TNF-α), in a mouse macrophage cell line, in a TLR5-dependent manner. The Gag/FliC expression construct was cloned into a bacterial artificial chromosome encoding the rhesus CMV (RhCMV) genome, and infectious RhCMV was generated following transfection of TeloRF cells. This virus stably expressed an SIV Gag/FliC fusion protein through four serial passages. Lysates generated from infected cells induced TNF-α in a TLR5-dependent manner. Western blot analysis of infected cell lysates verified expression of a Gag/FliC fusion protein using a SIV p27 capsid monoclonal antibody. Lastly, rhesus macaques inoculated with this novel RhCMV virus demonstrated increased inflammatory responses at the site of inoculation seven days post-infection when compared to the parental RhCMV. These results demonstrate that an artificially constructed replicating RhCMV expressing an SIV Gag/FliC fusion protein is capable of activating TLR5 in a macrophage cell line in vitro and induction of an altered inflammatory response in vivo. Ongoing animals studies are aimed at determining vaccine efficacy, including subsequent challenge with pathogenic SIV.

Aging and certain viral infections can negatively impact humoral responses in humans. To further develop the nonhuman primate (NHP) model for investigating B cell dynamics in human aging and infectious disease, a flow cytometric panel was developed to characterize circulating rhesus B cell subsets. Significant differences between human and macaque B cells included the proportions of cells within IgD+ and switched memory populations and a prominent CD21-CD27+ unswitched memory population detected only in macaques. We then utilized the expanded panel to analyze B cell alterations associated with aging and acute simian immunodeficiency virus (SIV) infection in the NHP model. In the aging study, distinct patterns of B cell subset frequencies were observed for macaques aged one to five years compared to those between ages 5 and 30 years. In the SIV infection study, B cell frequencies and absolute number were dramatically reduced following acute infection, but recovered within four weeks of infection. Thereafter, the frequencies of activated memory B cells progressively increased; these were significantly correlated with the magnitude of SIV-specific IgG responses, and coincided with impaired maturation of anti-SIV antibody avidity, as previously reported for HIV-1 infection. These observations further validate the NHP model for investigation of mechanisms responsible for B cells alterations associated with immunosenescence and infectious disease.

Breastfeeding is the gold standard for feeding infants because of its long-term benefits to health and development, but most infants in the United States are not exclusively breastfed in the first six months. We enrolled 24 infants who were either exclusively breastfed or supplemented with formula by the age of one month. We collected diet information, stool samples for evaluation of microbiotas by 16S rRNA sequencing, and blood samples for assessment of immune development by flow cytometry from birth to 6 months of age. We further typed the Bifidobacterium strains in stool samples whose 16S rRNA sequencing showed the presence of Bifidobacteriaceae. Supplementation with formula during breastfeeding transiently changed the composition of the gut microbiome, but the impact dissipated by six months of age. For example, Bifidobacterium longum, a bacterial species highly correlated with human milk consumption, was found to be significantly different only at 1 month of age but not at later time points. No immunologic differences were found to be associated with supplementation, including the development of T-cell subsets, B cells, or monocytes. These data suggest that early formula supplementation, given in addition to breast milk, has minimal lasting impact on the gut microbiome or immunity.