Pharmacokinetics of long-acting cephapirin and cloxacillin after intramammary administration in dairy goats.

Determining the pharmacokinetics of intramammary antimicrobials in goats can assist in predicting appropriate meat and milk withdrawal intervals for drugs that are effective at treating subclinical mastitis due to non-aureus Staphylococci during the dry period. Twenty-four healthy, lactating does were enrolled in this study. Half were administered 300 mg of cephapirin benzathine (ToMORROW, Boehringer Ingelheim Vetmedica, Duluth, GA) via intramammary infusion into each half of the udder. The remaining does had 500 mg cloxacillin benzathine (Orbenin DC, Merck & Co., Rahway, NJ) administered per half. Plasma was collected before treatment and for 7 days post-treatment followed by analysis via liquid chromatography with tandem mass spectroscopy. Pharmacokinetic parameters were determined using noncompartmental methods via commercial software (MonolixSuite). The mean maximum concentration (Cmax) of cephapirin of 0.073 µg/mL was noted at 7.06 h post-administration (Tmax). The area under the plasma concentration curve based on the final sampling point (AUClast) was 1.06 h × µg/mL. The mean residence time until the final sampling point (MRTlast) was 13.55 h. Mean terminal half-life (T½) of cephapirin was 6.98 h. In CLOX does, Cmax was 0.074 µg/mL with a Tmax of 18 h, AUClast was 5.71 h × µg/mL, T½ was 77.45 h, and MRTlast was 65.36 h. Despite both products being formulated with benzathine salts, marked differences were noted in pharmacokinetic parameters including AUC, T1/2, and MRTlast. This data will be used to plan sampling schedules for milk and tissue residue depletion studies for both products.

Canine Intestinal Organoids as a Novel In Vitro Model of Intestinal Drug Permeability: A Proof-of-Concept Study.

A key component of efforts to identify the biological and drug-specific aspects contributing to therapeutic failure or unexpected exposure-associated toxicity is the study of drug-intestinal barrier interactions. While methods supporting such assessments are widely described for human therapeutics, relatively little information is available for similar evaluations in support of veterinary pharmaceuticals. There is, therefore, a critical need to develop novel approaches for evaluating drug-gut interactions in veterinary medicine. Three-dimensional (3D) organoids can address these difficulties in a reasonably affordable system that circumvents the need for more invasive in vivo assays in live animals. However, a first step in developing such systems is understanding organoid interactions in a 2D monolayer. Given the importance of orally administered medications for meeting the therapeutic need of companion animals, we demonstrate growth conditions under which canine-colonoid-derived intestinal epithelial cells survive, mature, and differentiate into confluent cell systems with high monolayer integrity. We further examine the applicability of this canine-colonoid-derived 2D model to assess the permeability of three structurally diverse, passively absorbed ß-blockers (e.g., propranolol, metoprolol, and atenolol). Both the absorptive and secretive apparent permeability (Papp) of these drugs at two different pH conditions were evaluated in canine-colonoid-derived monolayers and compared with that of Caco-2 cells. This proof-of-concept study provides promising preliminary results with regard to the utility of canine-derived organoid monolayers for species-specific assessments of therapeutic drug passive permeability.

Extensive Evaluation of a Method for Quantitative Measurement of Aflatoxins B1 and M1 in Animal Urine Using High-Performance Liquid Chromatography with Fluorescence Detection.

BACKGROUND: Aflatoxins (AFs) are common feed contaminants and are one of the common causes of toxin-related pet food poisoning and recalls. OBJECTIVE: Currently, there are no validated methods for the detection and quantitation of AFs in biological matrices to diagnose AF exposure in live animals. Following a successful intra-laboratory method development to quantify AFB1 and AFM1 in animal urine by HPLC with fluorescence detection (HPLC-FLD), the present study was conducted to extensively evaluate the method performance in an unbiased manner using blinded samples. METHODS: The evaluation included two stages. First, the performance was verified in the method-originating laboratory in a single-laboratory blinded method test (BMT-S) trial followed by a multi-laboratory blinded method test (BMT-M) trial. RESULTS: In both trials, accuracy, repeatability, and reproducibility were satisfactory confirming the relatively good ruggedness and robustness of the method and ensuring that it will perform as expected if used by other laboratories in the future. CONCLUSIONS: We extensively evaluated the performance of a quantitative method to detect AFB1 and AFM1 in animal urine by HPLC-FLD by two different laboratories in two separate BMT-S and BMT-M trials. Both BMT results demonstrated the satisfactory accuracy and precision of the method. It is now available to be adopted by other diagnostic laboratories for purposes of diagnosing AF intoxication in animals. HIGHLIGHTS: A simple urine-based diagnostic test method using HPLC-FLD that originated in a single laboratory now has passed a multi-laboratory evaluation and is now available to be shared with other diagnostic laboratories for purposes of diagnosing AF intoxication in animals so better treatment can be rendered.

Enteropathy and bone marrow hypoplasia associated with presumptive albendazole toxicosis in a juvenile Boer goat.

Albendazole is a widely used anthelmintic drug that is labeled for the treatment of specific nematodes and flukes in ruminants. Albendazole is approved for the treatment of liver flukes in goats (10 mg/kg PO for a single dose), but is commonly used extra-label in situations in which parasite resistance is an issue. Albendazole toxicosis has been reported in pigeons, doves, alpacas, humans, dogs, and cats. Here we report an adverse event in a 6-mo-old goat associated with extra-label use of albendazole (35.7 mg/kg PO daily for 3 d). Clinicopathologic findings included severe diarrhea and death, with small intestinal crypt necrosis and dysplasia, and severe bone marrow hypoplasia. Microbial and molecular testing and transmission electron microscopy ruled out infectious organisms. The described pathologic changes are similar to those reported in other species that have experienced toxicosis associated with albendazole. To our knowledge, bone marrow and intestinal lesions associated with albendazole use in the goat have not been reported previously. Veterinarians should be aware of potential adverse events and toxicoses associated with anthelmintic drugs, especially as parasite resistance increases, and extra-label usage, and the use of such drugs without veterinary supervision, becomes more common.

Acute hemorrhage and death in calves following chlorophacinone exposure.

Three calves were submitted to the Iowa State University Veterinary Diagnostic Laboratory for diagnostic evaluation following an abrupt increase in morbidity and mortality in a calf herd associated with epistaxis and widespread hemorrhage. Each of the submitted calves had moderate-to-severe hemorrhage within various tissues and body cavities, including the thymus, subcutaneous region of the neck, mediastinum, lungs, pericardial sac, heart, spleen, perirenal fat, urinary bladder, and skeletal muscle, including the diaphragm. An anticoagulant rodenticide screen was performed on the livers of each calf. Significant concentrations of chlorophacinone were detected at 4.2, 3.6, and 2.9 ppm in liver. Multiple piles and an open pail of white powdery material were present within the facility in which the calves were housed and were identified as the sources of chlorophacinone. Acute hemorrhage and death occurred in fourteen 1.5-mo-old, crossbred calves following ingestion of the vitamin K antagonist chlorophacinone.

Variable accuracy, precision, and consistency of compounded famciclovir formulated for management of feline herpesvirus-1 in cats.

OBJECTIVE: To evaluate compounded famciclovir suspensions for accuracy, precision, and consistency in drug content. PROCEDURES: Two compounded famciclovir concentrations were evaluated (250 and 400 mg/mL, 30 preparations total from nine 503A compounding pharmacies) with U.S. Food and Drug Administration (FDA)-approved famciclovir tablets as control. Drug quantification via high-performance liquid chromatography (with famciclovir reference standard and pramipexole internal standard) was performed at 0, 14, and 28 days with concentrations of 90%-110% of labeled dose considered acceptable (US Pharmacopoeia standards). RESULTS: FDA-approved tablets from three different manufacturers were found to be accurate and precise with acceptable drug content. A significantly greater mean deviation from labeled content was noted for 400 mg/mL suspensions (-52.9%) compared to 250 mg/mL suspensions (-18.0%). When assessing time points separately, 15/63 (24%) samples of 250 mg/mL and 0/27 (0%) samples of 400 mg/mL suspensions met the acceptance standards. Coefficients of variation (CV) in drug content among pharmacy batches ranged from 0.5% to 29%, with 5/10 formulations having significantly lower CV% compared to control (decreased precision). Similarly, drug content changed over time (0-28 days) in all compounded formulations, with both downward and upward trends observed (variable consistency). CONCLUSIONS: Most compounded famciclovir formulations were inaccurate, imprecise, and inconsistent. FDA-approved famciclovir tablets may be preferred over compounded famciclovir formulations for the management of feline herpesvirus-1. If compounded famciclovir is used in practice, a concentration of 250 mg/mL is preferred over 400 mg/mL given the lower accuracy of the higher concentration.

Chemical and microbiological stability of diluted ceftazidime in three different solutions under three storage temperatures over a 28 day period.

BACKGROUND: Ceftazidime (CAZ) solutions are being used based on anecdotal reports for otitis externa complicated by multidrug-resistant Pseudomonas aeruginosa (MDR PA). The chemical and microbiological stability of these proposed compounded solutions have not been evaluated, and likely are affected by the diluent and storage duration or temperature. HYPOTHESIS/OBJECTIVES: Compounded CAZ solutions would show variable degradation dependent on diluent, time and temperature. The antimicrobial activity of the solutions would reflect changes in concentration and not alterations to the chemical compound. METHODS AND MATERIALS: Ceftazidime was compounded with 100 mL 0.9% sodium chloride (NA+CAZ), 118 mL Triz-EDTA Aqueous flush (TE+CAZ) and 125 mL Douxo Micellar Solution (MI+CAZ). Aliquots of the solutions were stored at 25°C, 4°C and -20°C for 28 days. High-performance liquid chromatography was used to analyse CAZ recovery from compounded solutions at weekly intervals. A modified broth dilution technique was utilised to assess minimum inhibitory concentration (MIC) to monitor antimicrobial activity against a reference PA strain. RESULTS: Temperature, duration of storage and diluent each had independent effects on the chemical stability of CAZ. CAZ concentrations decreased over time as well as with increased temperature. NA+CAZ solutions exhibited the least degradation compared to the other solutions. The MIC for PA was most consistent for NA+CAZ solutions regardless of storage temperature and duration of storage. CONCLUSIONS AND CLINICAL IMPORTANCE: Chemical and microbiological stability of compounded CAZ solutions varied by diluent, storage temperature and duration of storage. Dilution in NA resulted in the lowest variation in stability over 28 days when stored at refrigerated or frozen temperatures compared to other diluents.

High-performance liquid chromatography and Enzyme-Linked Immunosorbent Assay techniques for detection and quantification of aflatoxin B1 in feed samples: a comparative study.

OBJECTIVE: Comparison was done between high-performance liquid chromatography (HPLC) and a competitive enzyme-linked immunosorbent assay (ELISA) for detection and quantification of aflatoxin B1 (AFB1) in feed samples. The two procedures were standardized and validated before the actual experiment. Five concentrations (0, 5, 10, 20 and 30 ppb) of feed samples were used for both methods. For the HPLC technique, the samples were extracted in acetonitrile/water (90/10) solution, cleaned-up using solid phase extraction (SPE) column, and derivatized by water/trifluoroacetic acid/glacial acetic acid (35/10/5) solution before instrument analysis. The samples were extracted in 70% methanol for the ELISA technique. RESULTS: The two tests showed very strong linearity with correlation coefficient value of > 0.99 using standard solutions. The mean recovery rate was 92.42% (with relative standard deviation (RSD) of 5.97) and 75.64% (RSD = 34.88) for HPLC and ELISA, respectively. There was no statistically significant difference in recovery rate between the two methods. There was a positive correlation (r = 0.84) between them which indicated that the two techniques can be used to detect and quantify aflatoxin B1 in feed samples. However, there were variations among replicates for the ELISA method, which shows that this method is more applicable for screening purposes.

Evaluation of a Diagnostic Method to Quantify Aflatoxins B1 and M1 in Animal Liver by High-Performance Liquid Chromatography with Fluorescence Detection.

Background: Aflatoxins (AFs) are secondary metabolites of fungi and are one of the causes of toxin-related pet food recalls. An intralaboratory method was previously developed to quantify aflatoxin B1 (AFB1) and aflatoxin M1 (AFM1) in animal liver by HPLC with fluorescence detection. Objective: The aim of this study was to extensively evaluate the method performance with a single-laboratory blinded method test (BMT-S) and a multilaboratory blinded method test (BMT-M). Methods: Blinded tissue samples were prepared by a third-party laboratory and sent out to participating laboratories for both BMT-S and BMT-M. Results: In both tests, participants analyzed blinded samples prepared by an independent laboratory. In the BMT-S, accuracy ranged between 111 and 154% for AFB1 and 113 and 159% for AFM1 within the quantitation range of 0.1-0.5 ng/g. The HorRat values for repeatability ranged between 0.1 and 0.3 for AFB1 and 0.3 and 0.6 for AFM1. In the BMT-M, the interlaboratory accuracy ranged between 77 and 81% for AFB1 and 83 and 85% for AFM1 within the quantitation range of 0.2-10 ng/g. The HorRat values for reproducibility ranged between 0.4 and 0.7 for AFB1 and 0.4 and 0.9 for AFM1. Both recovery and reproducibility were acceptable. Conclusions: BMT-M evaluation demonstrated that the method was suitable for quantitation of aflatoxins B1 and M1 in animal liver between laboratories. Highlights: The BMT-S and BMT-M results demonstrated that the method is rugged and reproducible among the participating laboratories.

Intra-laboratory Development and Evaluation of a Quantitative Method for Measurement of Aflatoxins B1, M1 and Q1 in Animal Urine by High Performance Liquid Chromatography with Fluorescence Detection.

Mycotoxins negatively impact animal health. Aflatoxins (AFs) are the most common mycotoxins affecting both large and small animals and are a common cause of toxin-related pet food recalls. Definitive diagnosis of aflatoxicosis is constrained by a lack of validated ante-mortem analytical methods for detection and quantitation of AFs and their metabolites in biological specimens. Herein, we developed and evaluated a urine-based quantitative method for measurement of aflatoxin B1 (AFB1) and its metabolites aflatoxin M1 (AFM1) and aflatoxin Q1 (AFQ1) in animal urine. (Some of the results have been presented at 59th AAVLD conference, Greensboro, North Carolina, October 13-19th, 2016.) This method uses an immuno-affinity column for clean-up and pre-column derivatization followed by high performance liquid chromatography analysis with fluorescence detection. The method has high selectivity, recovery (>81%) and sensitivity with an instrument limit of detection of 0.20-1.02 pg; instrument limit of quantitation of 0.77-4.46 pg; and a method lower limit of quantitation of 0.30-2.5 ng/mL. The method has high accuracy, repeatability, and is rugged against minor changes. However, because of poor sensitivity of AFQ1 at low concentrations we recommend this method for quantitative determination of AFB1 and AFM1, and for qualitative measurement of AFQ1 in animal urine for diagnosis of aflatoxicosis.

Intralaboratory development and evaluation of a high-performance liquid chromatography-fluorescence method for detection and quantitation of aflatoxins M1, B1, B2, G1, and G2 in animal liver.

Aflatoxins are potent mycotoxins with effects that include hepatotoxicity, immunosuppression, and suppression of animal growth and production. The etiologic diagnosis of aflatoxicosis, which is largely based on analysis of contaminated feed matrices, has significant disadvantages given the fact that representative feed samples may not be available and feed-based test methods are not confirmatory of an etiologic diagnosis. A tissue-based analytical method for biomarkers of exposure would be valuable for confirmation of aflatoxicosis. We describe in-house development and evaluation of a high-performance liquid chromatographic method with fluorescence detection and precolumn derivatization for determination of aflatoxins M1, B1, B2, G1, and G2 in animal liver. The method demonstrates good selectivity for the tested aflatoxins in the liver matrix. The overall range was 0.03-0.10 ng/g for limit of detection and 0.09-0.18 ng/g for limit of quantitation. The correlation coefficient (R2) of calibration curves was >0.9978 for AFM1, 0.9995 for AFB1, 0.9986 for AFB2, 0.9983 for AFG1, and 0.9980 for AFG2 For fortification levels of 0.2-10 ng/g, repeatability was 10-18% for AFM1, 7-14% for AFB1, 5-14% for AFB2, 6-16% for AFG1, and 10-15% for AFG2 Recovery was 52-57% for AFM1, 54-62% for AFB1, 55-61% for AFB2, 57-67% for AFG1, and 61-65% for AFG2 There was no liver matrix effect found. The method is rugged against minor changes based on the selected factors. The results indicate that the proposed method is suitable for quantitative determination of aflatoxins M1, B1, B2, G1, and G2 in liver.

Improved Tissue-Based Analytical Test Methods for Orellanine, a Biomarker of Cortinarius Mushroom Intoxication.

Orellanine (OR) toxin is produced by mushrooms of the genus Cortinarius which grow in North America and in Europe. OR poisoning is characterized by severe oliguric acute renal failure, with a mortality rate of 10%-30%. Diagnosis of OR poisoning currently hinges on a history of ingestion of Cortinarius mushrooms and histopathology of renal biopsies. A key step in the diagnostic approach is analysis of tissues for OR. Currently, tissue-based analytical methods for OR are nonspecific and lack sensitivity. The objectives of this study were: (1) to develop definitive HPLC and LC-MS/MS tissue-based analytical methods for OR; and (2) to investigate toxicological effects of OR in mice. The HPLC limit of quantitation was 10 µg/g. For fortification levels of 15 µg/g to 50 µg/g OR in kidney, the relative standard deviation was between 1.3% and 9.8%, and accuracy was within 1.5% to 7.1%. A matrix-matched calibration curve was reproduced in this range with a correlation coefficient (r) of 0.97-0.99. The limit of detection was 20 ng/g for LC-MS/MS. In OR-injected mice, kidney OR concentrations were 97 ± 51 µg/g on Day 0 and 17 ± 1 µg/g on termination Day 3. Splenic and liver injuries were novel findings in this mouse model. The new tissue-based analytical tests will improve diagnosis of OR poisoning, while the mouse model has yielded new data advancing knowledge on OR-induced pathology. The new tissue-based analytical tests will improve diagnosis of OR poisoning, while the mouse model has yielded new data advancing knowledge on OR-induced pathology.

A novel orellanine containing mushroom Cortinarius armillatus.

Orellanine (3,3',4,4'-tetrahydroxy-2,2'-bipyridine-1,1'-dioxide) is a tetrahydroxylated di-N-oxidized bipyridine compound. The toxin, present in certain species of Cortinarius mushrooms, is structurally similar to herbicides Paraquat and Diquat. Cortinarius orellanus and Cortinarius rubellus are the major orellanine-containing mushrooms. Cortinarius mushrooms are widely reported in Europe where they have caused human poisoning and deaths through accidental ingestion of the poisonous species mistaken for the edible ones. In North America, Cortinarius orellanosus mushroom poisoning was recently reported to cause renal failure in a Michigan patient. Cortinarius mushroom poisoning is characterized by delayed acute renal failure, with some cases progressing to end-stage kidney disease. There is debate whether other Cortinarius mushroom contain orellanine or not, especially in North America. Currently, there are no veterinary diagnostic laboratories in North America with established test methods for detection and quantitation of orellanine. We have developed two diagnostic test methods based on HPLC and LC-MSMS for identification and quantitation of orellanine in mushrooms. Using these methods, we have identified Cortinarius armillatus as a novel orellanine-containing mushroom in North America. The mean toxin concentration of 145 ug/g was <1% of that of the more toxic C. rubellus. The HPLC method can detect orellanine at 17 µg g(-1) while the LC-MSMS method is almost 2000 times more sensitive and can detect orellanine at 30 ng g(-1). Both tests are quantitative, selective and are now available for veterinary diagnostic applications.

Ergot alkaloid intoxication in perennial ryegrass (Lolium perenne): an emerging animal health concern in Ireland?

Four primary mycotoxicosis have been reported in livestock caused by fungal infections of grasses or cereals by members of the Clavicipitaceae family. Ergotism (generally associated with grasses, rye, triticale and other grains) and fescue toxicosis (associated with tall fescue grass, Festuca arundinacea) are both caused by ergot alkaloids, and referred to as 'ergot alkaloid intoxication'. Ryegrass staggers (associated with perennial ryegrass Lolium perenne) is due to intoxication with an indole-diperpene, Lolitrem B, and metabolites. Fescue-associated oedema, recently described in Australia, may be associated with a pyrrolizidine alkaloid, N-acetyl norloline. Ergotism, caused by the fungus Claviceps purpurea, is visible and infects the outside of the plant seed. Fescue toxicosis and ryegrass staggers are caused by Neotyphodium coenophalium and N. lolii, respectively. Fescue-associated oedema has been associated with tall fescue varieties infected with a specific strain of N. coenophialum (AR542, Max P or Max Q). The name Neotyphodium refers to asexual derivatives of Epichloë spp., which have collectively been termed the epichloë fungi. These fungi exist symbiotically within the grass and are invisible to the naked eye. The primary toxicological effect of ergot alkaloid involves vasoconstriction and/or hypoprolactinaemia. Ingestion of ergot alkaloid by livestock can cause a range of effects, including poor weight gain, reduced fertility, hyperthermia, convulsions, gangrene of the extremities, and death. To date there are no published reports, either internationally or nationally, reporting ergot alkaloid intoxication specifically associated with perennial ryegrass endophytes. However, unpublished reports from the Irish Equine Centre have identified a potential emerging problem of ergot alkaloid intoxication with respect to equines and bovines, on primarily perennial ryegrass-based diets. Ergovaline has been isolated in varying concentrations in the herbage of a small number of equine and bovine farms where poor animal health and performance had been reported. Additionally, in some circumstances changes to the diet, where animals were fed primarily herbage, were sufficient to reverse adverse effects. Pending additional information, these results suggest that Irish farm advisors and veterinarians should be aware of the potential adverse role on animal health and performance of ergot alkaloids from perennial ryegrass infected with endophytic fungi.