Histamine is one of the most important and toxic biogenic amines which may be present in food and may cause food poisoning in humans when contained at a high level. It is produced during bacterial decarboxylation of histidine in fish muscles. The aim of the study was to investigate the presence of histamine in fish and fish products available in Poland during 2014–2018. A total of 421 samples of raw (248), smoked (107), canned (50), and marinated fish (16) were analysed by high-performance liquid chromatography with diode array detection. Histamine was detected in 14.1% samples of raw fish, 29% of smoked fish, 22% of canned fish and 93.8% of marinated fish in concentrations ranging from 3.4 to 156.4 mg/kg. Content of this amine above 100 mg/kg was found in four samples: raw Atlantic salmon, smoked European sprat and two samples of marinated Atlantic herring. The study showed that fish and fish products on the Polish market generally meet the food safety criteria for histamine and are safe for consumers.

In veterinary medicine, sensitive and specific markers of the early stages of renal failure still remain to be established. Podocytes could be a promising diagnostic tool in veterinary nephrology, especially in the differentiation of active pathological disease and glomerulopathies. Podocin is one of the robust proteins exploitable in detection of podocyturia. This article presents podocyte detection in urine for diagnostic purposes in veterinary medicine using a variety of methods. We describe the advantages and disadvantages of the immunohistochemical technique currently used, and of scanning microscopy, chromatography, and immunostaining. The identification of podocin-positive cells is a promising diagnostic tool in the detection of the early stages of glomerular basement membrane damage. The detection of renal failure prior to the occurrence of azotaemia is of high clinical importance from the clinical and scientific points of view.

Introduction: In the European Union the use of steroid growth promoters is prohibited under Council Directive 96/22/EC. For effective control of illegal use of natural steroids, highly sensitive analytical methods are required, because sex hormones can be present in very low concentrations in biological samples. The aim of the study was to develop a confirmatory method for the detection of testosterone in bovine serum at ppt level. Material and Methods: 17β-testosterone and internal standards of 17β-testosterone-d2 were extracted from serum samples with a mixture of tert-butyl methyl ether/petroleum ether and were directly analysed by an LC/MS/MS on QTRAP 5500 instrument with a TurboIon-Spray source operating in a positive ionisation mode. Chromatographic separation was achieved on the analytical column Inertsil® ODS-3 with an isocratic elution using mobile phase consisting of acetonitrile, methanol, and water. Method validation has been carried out in accordance with the Commission Decision 2002/657/EC. Results: The method was characterised by good recovery (82%) and precision (R.S.D 17 %). Decision limit (CCα) and detection capability (CCβ) was 0.05 μg L⁻¹ and 0.09 μg L⁻¹ respectively. The method met the criteria set out in Commission Decision 2002/657/EC for the purpose of confirmation in terms of retention time and ion ratio in the whole range of its application. Conclusions: The developed method is specific and sensitive, suitable for measuring the natural level of testosterone in blood of cattle and for use in routine control programme for the detection of this hormone in bovine serum.

OBJECTIVE To evaluate the plasma disposition of mycophenolic acid (MPA) and its derivatives MPA glucuronide and MPA glucoside after twice-daily infusions of mycophenolate mofetil (MMF) in healthy cats for 3 days and to assess the effect of MMF administration on peripheral blood mononuclear cell (PBMC) counts and CD4+-to-CD8+ ratios. ANIMALS 5 healthy adult cats. PROCEDURES MMF was administered to each cat (10 mg/kg, IV, q 12 h for 3 days). Each dose of MMF was diluted with 5% dextrose in water and then administered over a 2-hour period with a syringe pump. Blood samples were collected for analysis. A chromatographic method was used to quantitate concentrations of MPA and its metabolites. Effects of MMF on PBMC counts and CD4+-to-CD8+ ratios were assessed by use of flow cytometry. RESULTS All cats biotransformed MMF into MPA. The MPA area under the plasma concentration–time curve from 0 to 14 hours ranged from 14.6 to 37.6 mg·h/L and from 14.4 to 22.3 mg·h/L after the first and last infusion, respectively. Total number of PBMCs was reduced in 4 of 5 cats (mean ± SD reduction, 25.9 ± 15.8% and 26.7 ± 19.3%) at 24 and 48 hours after the end of the first infusion of MMF, respectively. CONCLUSIONS AND CLINICAL RELEVANCE Plasma disposition of MPA after twice-daily IV infusions for 3 days was variable in all cats. There were no remarkable changes in PBMC counts and CD4+-to-CD8+ ratios.

Aflatoxin (AF)-contaminated and fumonisin B1 (FB1)-contaminated (culture material from Fusarium moniliforme) diets were fed singly and in combination to growing cross-bred barrows. Six barrows (3 replicates of 2 each; mean body weight, 17.5 kg) per group were fed: 0 mg of AF and 0 mg of FB1/kg of feed (control); 2.5 mg of AF/kg of feed; 100 mg of FB1/kg of feed; or 2.5 mg of AF plus 100 mg of FB1/kg of feed for 35 days. The effects on production performance, serum biochemical, hematologic, immunologic, and pathologic measurements were evaluated. Body weight, gain, and feed consumption were significantly (P < 0.05) decreased by AF and AF plus FB1 diets. The FB1 diet decreased feed consumption, and although body weight was numerically decreased, it was not statistically significant. Aflatoxin increased serum gamma-glutamyltransferase (GGT) activity and total iron concentration and decreased urea nitrogen concentration and unsaturated iron-binding capacity. The FB1-alone diet increased serum GGT activity, whereas the AF plus FB1 diet increased serum aspartate transaminase, cholinesterase, alkaline phosphatase, and GGT activities, increased RBC count, triglycerides, and total iron concentrations, and decreased unsaturated iron-binding capacity and urea nitrogen concentration. For the most part, the effects of the AF plus FB1 diet on body weight and hematologic measurements could be considered additive. However, the effect of the AF plus FB1 diet on cholinesterase and alkaline phosphatase activities was greater than additive and was a synergistic response. One pig in the FB1-diet group and 2 pigs in the combination-diet group died. Postmortem lesions in pigs of the FB1-diet group consisted of ascites and increased liver weight. Observations at necropsy for pigs of the AF plus FB1-diet group consisted of hydrothorax, ascites, pulmonary edema, gastric erosions and ulceration, and increased liver and spleen weights. The AF diet increased relative liver weight and resulted in liver that was pale, rubbery, and resistant to cutting. Histologic lesions consisted of hepatic necrosis or degeneration, or both, with variable degrees of bile duct proliferation in barrows of the AF-diet groups. Renal tubular nephrosis was observed in barrows of the FB1 diet group, but this was not consistent in the AF plus FB1-diet group. Cell-mediated immunity, as measured by mitogen-induced lymphoblastogenic stimulation index, was decreased in barrows of the AF and FB1-diet groups, and values in barrows given the combination diet were significantly decreased from those in barrows given the single toxin diets. It was concluded that AF and FB1 (from culture material), singly or in combination, can adversely affect clinical performance, serum biochemical, hematologic, and immunologic values and induce lesions in growing barrows. For most of the variables we evaluated under our study conditions and dosages of toxins, measurements were affected more by the combination diet than by either single toxin diet, and the toxic responses could be described as additive or more than additive, particularly for induction of liver 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 high-performance liquid chromatography method was developed for determination of flunixin in bovine plasma. The extraction procedure was easily performed and made it possible to detect low concentrations of flunixin with high accuracy. The limit of quantitation was 7 ng/ml (relative standard deviation = 18%, n = 10). The analytic method permits processing of 60 samples/d. Flunixin, as well as the internal standard (diclofenac sodium), belong to the group of nonsteroidal anti-inflammatory drugs, which are known to have a high degree of binding to plasma proteins. Therefore, an evaluation of several buffer systems was undertaken to optimize analytic conditions. Cattle were given 2.2 mg of flunixin meglumine/kg of body weight. In experiment 1, single injections were administered IV to q cow and IM to 1 heifer (7 days apart), and pharmacokinetic variables were calculated. The IV data were best described by a two-compartment model. The half-life after single IV or IM administration was around 4.0 hours. In experiment 2, the decreasing flunixin concentration was determined after the last of either 4 IM injections daily (n = 3 cows) or 2 IM injections daily (n = 3 cows) administered during a 14-day postpartum period. The half-life, determined between 48 and 96 hours after the last dose, was approximately 26 hours in both groups, and flunixin could be detected in plasma up to 8 days, on average. The protein binding of flunixin was studied, using the method of equilibrium dialysis. Flunixin was found to have a high degree of protein binding (ie, 99.4 +/- 0.2%) at a flunixin concentration in plasma of 3 to micrograms/ml. Differences in protein binding between cattle were not found.

Growth of Pasteurella haemolytica A1 in RPMI 1640 medium containing 0.5% bovine serum albumin (BSA) for 2.5 hours enhanced culture supernatant leukotoxic activity [30,700 +/- 12,900 toxic units/ml, compared with leukotoxic activity of culture supernatants produced in RPMI 1640 medium alone (120 +/- 40 toxic units/ml)]. Gel filtration chromatography of the leukotoxic activity from RPMI 1640 medium supernatants in buffer containing 50 mM NaCl indicated a single leukotoxic activity peak (peak I) eluting near the gel resin molecular mass exclusion limit (estimated molecular mass of approx 8,000 kd). In contrast, culture supernatants produced in RPMI 1640 plus bovine serum albumin medium (RPMI + BSA) had peak I and 2 additional leukotoxic activity peaks (peaks II and III) with estimated molecular mass of approximately 80 and < 30 kd, respectively. All leukotoxic activity peaks were composed of approximately 100-kd molecular mass leukotoxin protomer, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting with a monoclonal antibody against leukotoxin. Subjecting culture supernatant leukotoxic activity produced in RPMI + BSA to gel filtration chromatography in buffer containing 500 mM NaCl or 6M urea resulted in detection of only a single leukotoxic activity peak with estimated approximate molecular mass of 250 and 800 kd, respectively. These findings suggest that P haemolytica exists as a high molecular mass aggregate with low leukotoxic activity which, in the presence of BSA, partially disaggregates to multiple toxin forms with enhanced leukotoxic activity. Some of these leukotoxin forms interact with dextran-based gel resins at low ionic strength.

Intestinal permeability was assessed in 12 healthy cats by use of a differential sugar absorption test. A 50-ml isotonic aqueous solution containing a combination of 1.8 g of the disaccharide lactulose and 1.7 g of the monosaccharide mannitol was administered to cats via nasogastric tube. Urine was collected after 6 hours, and all urine samples were analyzed the same day, using a gas-liquid chromatographic technique (GLC) and an enzymatic assay (ENZ). Median urinary recovery of lactulose was 0.27 and 0.54% determined by GLC and ENZ, respectively. Differences between these groups were statistically significant (P = 0.023), and correlation between assays was high (r = 0.94, P < 0.01). Median urinary recovery of mannitol was 1.93 and 2.09% for GLC and ENZ, respectively. There were no statistically significant differences between these groups and the correlation between assays was high (r = 0.85, P < 0.01). The median lactulose-to-mannitol ratio was 0.29, using GLC, and was 0.52, using ENZ. Correlation of these ratios was again high (r = 0.93, P < 0.01).

The antibiotic polymyxin B sulfate is a cationic polypeptide with a unique cyclical configuration and distinct cationic characteristics. In this investigation, polymyxin B was evaluated to determine its ability to prevent synthesis of lactic acid and tumor necrosis factor-alpha (TNF-alpha) by lipopolysaccharide-stimulated strain RAW 2647 macrophage-like cell populations. In this context, gradient concentrations of polymyxin B were formulated in the presence of fixed concentrations of lipopolysaccharide fractions from Escherichia coli (B4:0111), E. coli (J5), Klebsiella pneumoniae, Pseudomonas aeruginosa, Salmonella minnesota, and S. typhimurium (Re). Quantitation of TNF-alpha was established by the application of a tissue culture-based biological assay system, using the WEHI 164 clone 13 indicator cell line. Investigations also included evaluation of the ability of gradient concentrations of lipopolysaccharide fractions from E. coli (B4:0111), E. coli (J5), K. pneumoniae, P. aeruginosa, S. minnesota, and S. typhimurium (Re) to form a complex with polymyxin B. This was established through application of high-performance thin-layer chromatography techniques. On the basis of the known molecular characteristics of lipopolysaccharide, its lipid A-core subfractions, and polymyxin B, these results imply that cytoprotective properties of polymyxin B are attributable to direct interaction and subsequent complex formation. More specifically, the mechanism by which polymyxin B exerts affinity for lipopolysaccharide fractions is proposed to occur through attractive ionic interactions established between the cationic diaminobutyric acid residues of polymyxin B and the mono- or diphosphate group(s) of the lipid A-core moiety. It is highly probable that this molecular phenomenon is accompanied by hydrophobic interactions established between the terminal methyloctanoyl or methylheptanoyl groups of polymyxin B and the saturated carbon chains of the lipid A-core subfraction of lipopolysaccharide fractions.