A radioimmunoassay test for tetracyclines (Charm II) was compared with high-pressure liquid chromatography (HPLC) for detection of oxytetracycline (OTC) residues in milk samples from individual lactating cows. Oxytetracycline was administered by 1 of 3 routes (IV, IM, or intrauterine) to 21 lactating dairy cows. A total of 292 duplicate milk samples were collected from milkings before and through 156 hours after OTC administration. Concentration of OTC in these samples was determined by use of the Charm II test and an HPLC method with a lower limit of quantitation, approximately 2 ng of OTC/ml. Samples were also classified with respect to presence of OTC residues relative to the FDA safe concentration (less than or equal to 30 ng/ml), using the Charm II (by control point determination) and HPLC methods. There was a significant (P less than or equal to 0.05) difference between test methods in classification of milk samples with respect to presence or absence of OTC at the FDA safe concentration. A total of 48 of the 292 test results (16.4%) did not agree. Using the HPLC test results as the standard with which Charm II test results were compared, 47 false presumptive-violative test results and 1 false presumptive-nonviolative Charm II test result (a sample containing 31 ng of OTC/ml, as evaluated by HPLC) were obtained. The samples with false presumptive-violative Charm II results contained (less than or equal to 30 ng of OTC/ml, as evaluated by HPLC. In some respects, the Charm II test performed appropriately as a screening test to detect OTC residues in milk samples from individual cows. However, the tendency for the test to yield presumptive-violative test results at OTC concentrations lower than the FDA safe concentration (as evaluated by HPLC), suggests that caution should be exercised in using the test as the sole basis on which a decision is made to reject milk.

Introduction: The article presents a rapid and simple analytical procedure for determination of four tetracyclines (oxytetracycline, tetracycline, chlortetracycline, and doxycycline) in animal medicated feedingstuffs. Material and Methods: Two-gramme samples were extracted by a Na₂EDTA-McIlvaine buffer (pH 4)/methanol mixtures (40/60, v/v). The determination was achieved by liquid chromatography using a Zorbax Eclipse XDB C18 analytical column with mass spectrometer detection (LC-MS). Results: Recoveries of the antibiotics from spiked feed samples ranged from 78.2% to 113.5%. The LOD and LOQ for tetracyclines in feeds ranged from 2.8 to 4.2 and from 4.3 to 5.7 mg/kg, respectively. Conclusion: The method was successfully validated and proved to be efficient, precise, and useful for quantification of tetracyclines in medicated feedingstuffs.

Multi-class and multi-residue analyses are very complex procedures because of the physico-chemical properties of veterinary drug residues and other contaminants. The purpose of the study was to develop an analytical method for the sensitive determination of 69 analytes in bovine milk by liquid chromatography electrospray ionisation–tandem mass spectrometry. Antimicrobial, anabolic hormone, lactone, β-agonist, mycotoxin and pesticide residues were analysed in 120 raw milk samples from different dairy farms in North Macedonia. Stable isotopically labelled internal standards were used to facilitate effective quantification of the analytes. The linear regression coefficients were higher than 0.99, the limits of detection ranged from 0.0036 to 47.94 μg/L, and the limits of quantification ranged from 0.053 to 59.43 μg/L. The decision limit values ranged from 0.062 to 211.32 μg/L and the detection capability from 0.080 to 233.71 μg/L. Average recoveries of the analytes spiked in raw milk were in the range of 70.83% to 109%, intra-day coefficient of variation (CV) values from 2.41% to 22.29%, and inter-day CV values from 3.48% to 23.91%. The method was successfully applied in the testing of bovine milk samples. In five samples residues were detected. They were sulfadimethoxine (in two samples), enrofloxacin, tetracycline and oxytetracycline and were at concentrations below the EU maximum residue limit. The method is useful for routine testing for this group of chemical hazards in bovine milk.

The aim of this study was to present two outbreaks of bovine abortion due to Leptospira infection in cattle herds located in the northern part of Sicily (Italy). The animals were positive for Leptospira interrogans serogroup Sejroe serovar Hardjo in a microscopic agglutination test (MAT). A total of 23 Charolaise cows (farm A) and 75 Limousine bulls and Cinisara and Modicana cows (farm B) were enrolled in this study. The blood samples were collected from all subjects at the following time points: before a cycle of intramuscular treatment with oxytetracycline dihydrate (T0), after 5–6 weeks from the treatment (T1), and every 10 weeks until seronegativisation (T2 in Farm A and T3 in Farm B). A serological test (MAT) was used for the diagnosis of leptospirosis. Two samples from farm A (2/23) and 29 samples from farm B (29/75) were positive to Leptospira interrogans, serogroup Sejroe, serovar Hardjo in the MAT. Leptospira spp. DNA was detected by real-time PCR in the urine sample of one positive cow on farm A, and in placenta and brain samples belonging to one aborted foetus on farm B. It is important to use serological and molecular diagnostic techniques complementarily to identify infected individuals.

OBJECTIVE To identify clinical or clinicopathologic variables that can be used to predict a positive PCR assay result for Anaplasma phagocytophilum infection in equids. ANIMALS 162 equids. PROCEDURES Medical records were reviewed to identify equids that underwent testing for evidence of A phagocytophilum infection by PCR assay between June 1, 2007, and December 31, 2015. For each equid that tested positive (case equid), 2 time-matched equids that tested negative for the organism (control equids) were identified. Data collected included age, sex, breed, geographic location (residence at the time of testing), physical examination findings, and CBC and plasma biochemical analysis results. Potential predictor variables were analyzed by stepwise logistic regression followed by classification and regression tree analysis. Generalized additive models were used to evaluate identified predictors of a positive test result for A phagocytophilum. RESULTS Total lymphocyte count, plasma total bilirubin concentration, plasma sodium concentration, and geographic latitude were linear predictors of a positive PCR assay result for A phagocytophilum. Plasma creatine kinase activity was a nonlinear predictor of a positive result. CONCLUSIONS AND CLINICAL RELEVANCE Assessment of predictors identified in this study may help veterinarians identify equids that could benefit from early treatment for anaplasmosis while definitive test results are pending. This information may also help to prevent unnecessary administration of oxytetracycline to equids that are unlikely to test positive for the disease.

Milk antimicrobial residues are a serious concern for the dairy industry. Residues of the tetracycline family of antimicrobials have been reported in market milk by investigators, using radioimmunoassay and microbial receptor technology (hereafter referred to as the Charm II test). In response to these reports, an investigation was conducted to determine the potential of 3 extra-label routes of oxytetracycline (OTC) administration to cause milk residues above the Food and Drug Administration safe value of 30 parts per billion (ppb). Lactating Holstein cows were administered OTC once by use of 1 of 3 routes: IV at 16.5 mg/kg of body weight (n = 6); IM at 11 mg/kg (n = 6); and intrauterine (IU) at 2 g in 500 ml of saline solution/cow (n = 6). Duplicate milk samples were collected at the milking prior to drug administration and for the next 13 milkings at 12-hour intervals. Concentrations of OTC in milk samples were analyzed by use of the Charm II test for tetracyclines (limit of OTC detection, approx 5 ppb) and were compared with concentrations determined by use of a high-performance liquid chromatography (HPLC) method (lower limit of OTC quantitation, approx 2 ppb). The potential for milk OTC residues above the Food and Drug Administration safe value of 30 ppb after treatment was considerably greater for the IV and IM routes, compared with the IU route. Mean peak OTC concentrations in milk at the first milking after treatment for the HPLC and Charm II tests were approximately 3,700 to 4,200 ppb for the IV route, 2,200 to 2,600 ppb for the IM route, and 186 to 192 ppb for the IU route, respectively. Pharmacokinetic analysis, based on milk OTC concentrations, indicated that the area under the curve (AUC) and milk maximal concentration (Cmax) differed significantly (P < 0.001) among routes of administration. The AUC was similar for IV and IM administrations; values for both were greater than the AUC for IU administration. The Cmax was greatest for IV, intermediate for IM, and least for IU administration. There were significant (P less than or equal to 0.01) differences in AUC between assay methods (Charm II vs HPLC) for the IV route. Concentrations of OTC in milk determined by the Charm II test were often greater than those determined by HPLC. Administration of OTC to lactating cows via these routes is extra-label drug use. Failure to withhold the product from early milkings of cows administered OTC by the IV or IM route should be considered a potential cause of OTC residues in market milk. Milk from nearly all cows contained OTC (< 30 ppb), the Food and Drug Administration safe level, by 120 hours after OTC administration. Use of appropriate withholding times and antibiotic residue testing is indicated to avoid OTC residues.

A clinical trial examining the efficacy of 2 drugs for treatment of a natural epizootic of infectious bovine keratoconjunctivitis was performed. The study was conducted in 103 grazing Hereford calves during the summer of 1985. The calves were prospectively and randomly assigned to 1 of 3 groups at the beginning of the study on June 17, and were examined 3 times weekly thereafter until the final observation on August 6. Calves in group 1 (n = 34) were not treated and were used as controls. Calves of group 2 (n = 34) with corneal ulcers were treated with a long-acting oxytetracycline formulation (OTC group). The parenteral treatment was repeated in 72 hours. Affected calves of group 3 (n = 35) were treated topically with furazolidone spray when they developed new corneal ulcers, or when existing lesions worsened during subsequent examination periods (NFZ group). Healing times of the corneal ulcers were reported in 3 ways: the combined times for ulcers present in both eyes of a calf simultaneously (method A), independent times of each ulcer on a calf (method B), and time of the first ulcer for each calf (method C). Censored healing times were examined as left censored (ulcer present at the beginning of the study), right censored (ulcer not healed at the end of the study), or uncensored (true) healing times. The effect that the treatments had on healing times were investigated by use of notched box and whisker plots, life tables, and Cox regression models. The analysis indicated that treatment of calves with either antimicrobial reduced the healing time of corneal ulcers, compared with untreated controls. Calves treated with OTC had shorter periods with ulcers present on both eyes than did NFZ-treated calves. The healing time of the first ulcer on a calf was faster when treated with either antimicrobial than when not treated, but no significant difference between periods for OTC and NFZ treatments was found. Censored healing times were consistently longer than uncensored healing times. Box and whisker plots indicated that both treatments shortened healing times more than those for controls, and OTC shortened healing times more than did NFZ for responses A and B (but not C). Life tables showed that OTC healing times were shorter than those for controls, and NFZ shorter than controls for response B and C (but not A). Cox regression model (for response A) showed a borderline significant difference between times for OTC group and controls, and no significant difference between times for NFZ group and controls.

A 2 X 2 crossover design trial was conducted in gilts to determine the bioavailability and pharmacokinectics of tetracycline hydrochloride. The bioavailability of tretracycline hydrochloride administered orally to fasted gilts was approximately 23%. After intravascular administration, the disposition kinetics of tetracycline in plasma were best described by a triexponential equation. The drug had a rapid distribution phase followed by a relatively slow elimination phase, with half-life of 16 hours. Its large volume of distribution (4.5 +/- 1.06 L/kg) suggested that tetracycline is distributed widely in swine tissues. Total body clearance was 0.185 +/- 0.24 L/kg/h. Other pharamacokinectic variables were estimated. In a second trial, 3 gilts were fed a ration containing 0.55 g of tetracycline hydrochloride/kg of feed. Resulting plasma concentration of tetracycline was determined at selected times during 96 hours after exposure to the medicated feed. Plasma drug concentration peaked (0.6 microgram/ml) at 72 hours after access to the medicated feed.

OBJECTIVE To evaluate IM injection of oxytetracycline as an experimental model to induce pain and assess the analgesic efficacy of flunixin meglumine (FM) in dairy cows. ANIMALS 15 healthy nonlactating Jersey (n = 10) and Holstein (5) cows. PROCEDURES In the first of 2 experiments, 5 Jerseys were administered oxytetracycline (10 mg/kg, IM), divided between the right side of the neck and left hind limb. The left side of the neck and right hind limb received sham injections. Cows were also randomly assigned to receive FM (2.2 mg/kg, IV; n = 3) or an equal volume of saline (0.9% NaCl) solution (0.044 mL/kg, IV; control; 2) once daily for 5 days. The mechanical nociceptive threshold (MNT) was measured before oxytetracycline administration and at predetermined times after each injection of the assigned treatment. Experiment 2 was similar to experiment 1 except it involved 5 Jerseys and 5 Holsteins, oxytetracycline was injected only in a hind limb, and the assigned treatment was administered for 10 days. RESULTS For both experiments, mean MNT for the oxytetracycline injection site was consistently less than that for the sham injection site in the hind limbs, and mean MNT at the hind limb oxytetracycline injection site for FM-treated cows was greater than that for control cows beginning on day 3. CONCLUSIONS AND CLINICAL RELEVANCE IM injection of oxytetracycline in a hind limb reliably induced signs of pain in dairy cows and, with validation, might be useful as an experimental model for assessing pain mitigation strategies in cattle.