The use of more stringent penicillin detection methods in edible tissues at processing and a zero tolerance for the presence of detectable residues in some territories has resulted in an increase in penicillin residue violations at slaughter and caused pork producers to explore alternative antimicrobial options. A growing number of swine producers are utilizing ampicillin in place of penicillin in treatment regimens. This study investigated the tissue residues of ampicillin, an aminopenicillin used in swine for the treatment of gram positive and selected gram negative aerobic and anaerobic bacterial infections. The study also examined the ability to predict tissue residue levels using antemortem sample types including plasma, oral fluids, and urine. Furthermore, the project explored the ability of bench top real-time tests including the kidney inhibition swab (KIS) test, SNAP test kit, and the CHARM test to detect ampicillin levels in tissues, oral fluids, and urine, and compared those results with the detection of ampicillin in the same samples using high-performance liquid chromatography- mass spectrometry (HPLC-MS).

Sixty (60) cull sows were randomly assigned to two treatment groups (n= 30 sows/ group). Treatment Group 1 (TG1) was administered a dose of 6 mg/kg ampicillin trihydrate and Treatment Group 2 (TG2) was administered a dose of 12 mg/kg ampicillin trihydrate once daily for three days. Each treatment group was then divided into ten (10) groups of three (3) sows/ group, each group corresponding to a necropsy timepoint at 1, 3, 5, 8, 10, 12, 15, 20, 30, and 40 days post-administration of the final ampicillin trihydrate administration. Blood samples and oral fluids were collected from all sows immediately before first administration of ampicillin and immediately before euthanasia on sows to be necropsied. Urine and tissue samples (liver, kidney, injection site, skeletal muscle) were collected at necropsy.

A tolerance of 10 ng/g is established for negligible residues of ampicillin in the uncooked edible tissues of swine and cattle. Ampicillin tissue residues were detectable above the tolerance in the kidney at one day post-administration of ampicillin in both treatment groups. Tissue residues in the injection sites were above the tolerance in all groups out to 40 days after treatment. Presence of this residue may be due to a thick layer of fat over the injection site that may reduce drug absorption creating a drug depot. Ampicillin that may have been injected into this fat layer would likely have a decreased elimination compared to injection in the muscle and result in prolonged residues. Plasma concentrations of ampicillin closely approximated the kidney residue profile. All sows sampled one day post-administration of the last dose of ampicillin had detectable concentrations of ampicillin in the plasma. One sow in TG2 had plasma ampicillin concentrations at 3 days post-administration. A longer ampicillin residue was observed in the urine samples. In both treatment groups, ampicillin was found in urine up to 10 days post-administration. Urine is the primary route of excretion of beta-lactam antimicrobials. This combined with difference in the extraction procedures between urine and tissues could result in detectable levels of ampicillin accumulating in the urine when it could not be detected in kidney tissue. No ampicillin was detected in any of the oral fluid samples. Comparing the LC-MS results to the KIS test on kidney tissue residues, only one positive and one uncertain sample for ampicillin were found, both in TG1 at one day post-administration. This result shows the higher sensitivity of the LC-MS test for residue determination compared to the KIS test. SNAP test results showed very little association to ampicillin residues in urine and oral fluids detected by LC-MS. The SNAP test on urine indicated all groups of sows out to 30 days post-administration were positive for ampicillin residues. SNAP test on oral fluids also had no correlation with LC-MS values.
There was a statistically significant association between the presence of ampicillin in the kidney and a positive urine CHARM and LCMS test. There was almost perfect agreement between the results of the LCMS test on urine and the CHARM urine test. There was only fair agreement between the SNAP test and the LCMS urine test. There was poor agreement between the LCMS urine test and the KIS test on the kidney. Furthermore, there was only fair agreement between the LCMS test on the kidney tissue and the KIS test.
Tissue residues for ampicillin can be found in kidney tissues at one day following ampicillin treatment. Tissue residues for ampicillin at the injection site are seen at least up to 40 days following treatment. Sows appear to be able to metabolize ampicillin rapidly, with the exception of ampicillin at the site of injection. According to the recommended 15-day withdrawal on ampicillin, no detectable levels should be found in kidneys at this 15 day recommendation. Increasing needle length to ensure ampicillin injection only in muscle may reduce the persistent injection site residues. Plasma concentrations correlate closely with kidney tissue concentrations and may be an appropriate sample for residue testing before slaughter. No differences were observed in tissue residues seen in TG1 or TG2. Plasma samples had ampicillin concentrations at 3 days post-administration in TG2 (12 mg/kg), but only 1 day in TG1. Other sample types showed no difference in the two dosages. Oral fluids appear to be less reliable methods for ante-mortem residue level testing as ampicillin was not detected in this matrix, even in the presence of tissue residues. Benchtop tests like the KIS, SNAP, or CHARM are less accurate methods of residue determination than more sensitive methods like the LC-MS.
Contact information: Dr. Johann F. Coetzee, Veterinary Diagnostic and Production Animal Medicine, College of Veterinary Medicine, Iowa State University, Ames, Iowa, USA, 50011; Tel.: + 1 515-294-7424; fax: + 1 515 294-1072; E-mail address: [email protected]