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  • Author or Editor: James A. Havel x
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Objective—To determine the effects of protease inhibitors and holding times and temperatures before processing on the stability of substance P in bovine blood samples.

Samples—Blood samples obtained from a healthy 6-month-old calf.

Procedures—Blood samples were dispensed into tubes containing exogenous substance P and 1 of 6 degradative enzyme inhibitor treatments: heparin, EDTA, EDTA with 1 of 2 concentrations of aprotinin, or EDTA with 1 of 2 concentrations of a commercially available protease inhibitor cocktail. Plasma was harvested immediately following collection or after 1, 3, 6, 12, or 24 hours of holding at ambient (20.3° to 25.4°C) or ice bath temperatures. Total substance P immunoreactivity was determined with an ELISA; concentrations of the substance P parent molecule, a metabolite composed of the 9 terminal amino acids, and a metabolite composed of the 5 terminal amino acids were determined with liquid chromatography–tandem mass spectrometry.

Results—Regarding blood samples processed immediately, no significant differences in substance P concentrations or immunoreactivity were detected among enzyme inhibitor treatments. In blood samples processed at 1 hour of holding, substance P parent molecule concentration was significantly lower for ambient temperature versus ice bath temperature holding conditions; aprotinin was the most effective inhibitor of substance P degradation at the ice bath temperature. The ELISA substance P immunoreactivity was typically lower for blood samples with heparin versus samples with other inhibitors processed at 1 hour of holding in either temperature condition.

Conclusions and Clinical Relevance—Results suggested that blood samples should be chilled and plasma harvested within 1 hour after collection to prevent substance P degradation.

Full access
in American Journal of Veterinary Research


Objective—To evaluate the impact of oxytetracycline exposure on horizontal transfer of an antimicrobial resistance plasmid.

Sample—Populations of Salmonella enterica subsp enterica serovar Typhimurium and Escherichia coli.

Procedures—Mixed populations of plasmid donor (Salmonella Typhimurium) and recipient (E coli) bacteria were assigned to 1 of 2 simulated oxytetracycline dosing regimens (high peak concentration-short elimination half-life [HC-SHL] or low peak concentration—long elimination half-life [LC-LHL]) or served as untreated control replicates. Donor, recipient, and transconjugant (E coli that acquired the plasmid) bacteria populations were quantified at 12, 24, and 36 hours after oxytetracycline administration by use of culture on selective bacterial growth media.

Results—The ratio of transconjugant to donor bacteria was significantly reduced in the oxytetracycline-exposed replicates, compared with the ratio for the control replicates, at 12 hours. At 24 and 36 hours, results for the HC-SHL regimens were not significantly different from results for the respective control replicates, and results for the LC-LHL regimens also were not significantly different from results for the respective control replicates. The oxytetracycline concentration at these time points (12 hours in the HC-SHL regimen and all 3 time points in the LC-LHL regimen) were in excess of the minimum inhibitory concentration of the recipient bacteria.

Conclusions and Clinical Relevance—Transfer of antimicrobial resistance plasmids may be suppressed in vitro by oxytetracycline exposure at concentrations greater than the minimum inhibitory concentration of the recipient bacteria.

Full access
in American Journal of Veterinary Research