Using roquefortine C as a biomarker for penitrem A intoxication in a beef herd

Fifteen grazing beef cattle and calves presented a history of neurological signs like ataxia, intentional head tremors, muscle twitching. Nervous ketosis, nervous BVD, BHV-1,5, tremorgenic intoxication from hay, and Listeriosis were considered as differential diagnosis. Blood samples were collected. Inspection of hay bales showed large white dusty and moldy areas. Samples were taken and analyzed. Altered hay was immediately removed in all animals’ stock. No alterations were found in blood tests. Food analysis showed high concentrations of Roquefortine C (RC) (345 μg/kg DM). Tremorgenic syndrome has been reported in Penitrem A (PA) intoxication, but PA is difficult to isolate in laboratory conditions. Both RC and PA are produced by Penicillum spp. RC has been associated with PA in tremorgenic toxicosis in dogs and it might be considered a valuable diagnostic marker for PA intoxication. The neurological signs were due to tremorgenic intoxication after feeding of spoiled forage contaminated with mycotoxines

Clinical utility of urine kidney injury molecule-1 (KIM-1) and gamma-glutamyl transferase (GGT) in the diagnosis of canine acute kidney injury

The aim of the present study was to evaluate the sensitivity and specificity of urine KIM-1 and urine GGT for the detection of naturally-occurring AKI, compared to healthy control dogs, dogs with stable chronic kidney disease (CKD), and dogs with lower urinary tract disorders (LUTD). The study included AKI grade 1 (n = 21), AKI grade 2 to 5 (n = 11), stable CKD (n = 11), LUTD (n = 15), and healthy dogs (n = 37). Urine KIM-1 (ng/mg) and GGT (U/l) were normalized to urine creatinine (uCr). Statistically significant difference in KIM/uCr (p = 0.0007) and GGT/uCr (p < 0.0001) was found among the study groups. Area under the curve (AUC) for KIM-1/uCr and GGT/uCr as predictors of AKI was 0.81 and 0.91 respectively. Values of KIM-1/uCr of 0.73 ng/mg and of GGT/uCr of 54.33 showed the best combination of sensitivity and specificity (75% and 75.6%; 85.7% and 89.1% respectively). A significant positive correlation (p < 0.0001) between KIM-1/uCr and GGT/uCr was found. Both urine KIM-1/uCr and GGT/uCr seemed to be potentially good markers for the diagnosis of AKI. Dogs with AKI showed significantly higher levels of urine KIM-1/uCr and urine GGT/uCr, compared with healthy dogs. Caution should be used in the evaluation of elevated urine KIM-1/uCr and GGT/uCr in dogs with pre-existing CKD and/or LUTD. Urine KIM-1/uCr and GGT/uCr might have a significant clinical utility, as complementary test, particularly in diagnosis early, non-azotemic stages of AKI

DETERMINATION OF OCHRATOXIN A IN FARMED FISH BY ENZYMATIC DIGESTION (ED) COUPLED TO HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY WITH A FLUORESCENCE DETECTOR (HPLC-FLD)

Several studies have demonstrated that fish feeds contain significant concentrations of chemical contaminants, many of which can bioaccumulate and bioconcentrate in fish tissues (1).  The serious concern regarding the use of fish meal and fish oil in the the aquaculture industry has led to extensive search of alternative raw materials for aquafeeds. The most obvious alternatives are oils and proteins of plants origin. The use of these alternative feed ingredients can introduce contaminants that were previously not associated with fish farming such as mycotoxins (2). Ochratoxin A (OTA) is a mycotoxin produced as a secondary metabolite by various Aspergillus and Penicillium species with nephrotoxic, carcinogenic, immunotoxic and teratogenic potential (3). OTA has been found in several food commodities, including cereals and can also be present in food of animal origin as a result of carryover from contaminated feed (3). The aim of the present study was to determine OTA concentrations in muscle, kidney and liver of 10 seabream and 10 seabass of farmed origin collected on the market. Analysis will be performed by using an enzymatic digestion (ED) method coupled to high-performance liquid chromatography with a fluorescence detector (HPLC-FLD). Fish tissues were digested for 1 hour at 37°C with a 1% pancreatin solution in a phosphate buffer and then cleaned up with ethylacetate. After being evaporated to dryness and re-dissolved, the sample was processed using HPLC-FLD. The method was validated for: specificity, recovery, trueness, selectivity, linearity, limit of detection (LOD) and limit of quntification (LOQ), repeatability and reproducibility. Recoveries of analytical method were higher than 85 % for all the matrices. Intra and inter-day repeatability expressed as relative standard deviation were less than 9%. The LOD and LOQ for liver and muscles samples were 0.001 and 0.002 μg/kg, respectively. The LOD and LOQ for kidney samples were 0.01 and 0.02 μg/kg, respectively. The highest concentrations of OTA were found in kidney of the 20 fishes analyzed (rang

DEVELOPMENT OF AN ELECTROCHEMICAL SENSOR BASED ON SCREEN-PRINTED ELECTRODES FOR OCHRATOXIN A IN PORK MEAT SAMPLES

Ochratoxin A (OTA) is a nephrotoxic, immunosuppressive and teratogenic mycotoxin produced by As- pergillus and Penicillium spp. fungi during food storage. OTA can be detected in cereal products, coffee, wine, beer, cheese and in poultry and pork meat. Many detection techniques, such as liquid chromatography coupled with immunoaffinity column or solid phase extraction cleanup, have been used for OTA determina- tion in different samples (1). In recent years electrochemical techniques have been used for the rapid and accurate detection of OTA (2). The aim of the present study was to develop a new analytical method for OTA quantitative detection in pork meat based on electrochemical sensing, using graphite-based screen-printed electrodes and differential pulse voltammetry (DPV) as detection technique. Experiments were performed with an electrochemical transducer Palmsens, monitored with a personal computer using PSTrace software (Palm Instrument BV, Houten, The Netherlands) for data acquisition and subsequent analysis. The electrochemical assays were performed with miniaturized disposable graphite based screen-printed electrodes (EcoBioServices & Researches s.r.l., Florence, Italy). The effect of pH (range 2-7) and of ionic strength (KCl concentration range 10-200 mM) of the supporting electrolyte solution (acetate buffer) on the DPV peak current and potentials was investigated to optimize the DPV method. The effect of the DPV parameters on OTA oxidation peak was studied. Potential pulse amplitude (Epulse) was evaluated in the range of 10-100 mV. Step height was evaluated in the range of 2-10 mV. The influence of the scan rate was examined in the range of 0.005-0.1V/s. Standard addition method was applied for quantitative analysis. The method was applied for OTA determination in spiked pork meat samples. Results were compared with those provided by a reference HPLC method. The OTA peak current increased with increasing acetate buffer pH (from 2.0 to 7), thus pH of 7.0 for the supporting electrolyte solution was chosen. Concentrations of 75 mM KCl in the supporting electrolyte was selected. The optimization of DPV parameters indicated that best results for voltammograms were obtained from 0 to 1.1 V by using 5 mV potential step, 50 mV potential pulse, 0.01 V/sec scan rate and 0.07 sec time pulse; each scan was performed after an equilibrium time of 30 sec. Calibration graphs of peak height against concentration for OTA by DPV were plotted over the range 25-1000 μg/l in the supporting electrolyte with a LOQ of 25 μg/l. The findings obtained with voltammetric-based sensing were in good agreement with results obtained by HPLC analysis but matrix effects have been detected at lower OTA concentrations indicating the need of more selective extraction procedure. The proposed method is more rapid and inexpensive in comparison with the classical methods for OTA analysis, and can be considered a promising alternative for the evaluation of OTA in meat. 1) Turner et al, Anal Chim Acta, 2009, 632 ,168-180. 2) Prieto-Simón et al, TrAC, 2007, 26, 689-702

A possible tremorgenic mycotoxicosis by Roquefortine C in a bovine herd

A total of 15 beef cows and calves were referred for history of neurological signs. The animals (12/15 Chianina breed, 3/15 Limousine) were grazing in 300 ha area, fed with grass and hay. Inspection of the hay reveled macroscopic alterations, consisting of diffuse and heavy mold contamination of many hay bales. Due to the not cooperative attitude, the animals were only visually examined in the field; the neurological signs observed were ataxia, intentional head tremors and muscle twitching. Only 3 calves with severe neurological signs were housed in a medication area and underwent a complete clinical exam. All 3 calves showed intentional head tremors and muscle twitching; 1/3 presented severe ataxia and stiffness gait, while 2/3 calves were recumbent and unable to rise. The most important clinical data were: hyperthermia, tachypnea, tachycardia and long capillary refill time. The neurological examination showed deficits of V and VII cranial nerves. Calves could swallow, but they were unable to grab the food. Based on history and clinical examination the following differential diagnoses were considered: tremorgenic mycotoxicosis, nervous ketosis, nervous BVD form, BHV1-5, Listeriosis and WMD. Blood samples were collected for CBC count and biochemistry panel (TP, urea, creatinine, total and direct bilirubin, GGT, AST, CPK, Mg, Se and vit E), urinalysis was performed for ketone bodies. Calves were also tested for infectious diseases (Listeriosis, BVD, BHV 1-5). Multiple samples of altered hay were analyzed for mycotoxins and hay balls were removed in all animals’ stock. The grazing animals recovered spontaneously within 1 week along with 2/3 hospitalized calves, while 1/3 calf was euthanized due to poor general conditions. CBC, biochemistry panel, vit E and oligo-minerals resulted within normal ranges and no positivity for infectious agents were detected. Food analysis showed high concentrations of roquefortine C (RC): 345 μg/kg DM. Presence of RC in livestock food is highly reported, in particular in visibly moldy areas (1). RC intoxication causes anorexia, paralysis and several reports attribute it neurotoxic properties (2). In mice experimental intoxications induced muscle contractions, ataxia, prostration and intermittent seizures. RC intoxication, resembling penitrem A (PA) intoxication, has been reported in dogs. Moreover, RC is considered a sensitive biomarker for PA exposure. PA is a tremorgenic fungal toxin which intoxication causes ataxia, tachypnea, and sustained tremors. The pathophysiological mechanism by which mycotoxins affect the CNS is unknown but the biochemical lesions are reversible. Diagnosis is based on the clinical signs, demonstration of the mycotoxins in the feed and identification of the fungal elements in blood and feces. Affected animals recover completely when they are removed from infected pastures. Based on neurological signs, recovery after altered food removing and results of food analysis, the diagnosis of tremorgenic intoxication was hypothesized. Limits of this report are: lack of PA dosage in the food and lack of RC and PA evaluation in blood and feces of affected animals

Determination of ochratoxin A in pig tissues using enzymatic digestion coupled with high-performance liquid chromatography with a fluorescence detector

We present a new method for the rapid analysis of ochratoxin A (OTA) in pig tissues (muscle, liver and kidney) using enzymatic digestion (ED) coupled to high-performance liquid chromatography with a fluorescence detector (HPLC-FLD). OTA was digested with a 1% pancreatin solution in a phosphate buffer and then cleaned with ethylacetate. After being evaporated to dryness and re-dissolved, the sample was determined using HPLC-FLD. The method was validated taking into account the currently permitted limit of 1 μg/kg OTA in pork meat and derived products in Italy. The recovery was higher than 90%. Intra- and inter-day repeatability expressed as RSD were less than 7%. The LOD and LOQ were 0.001 and 0.002 μg/kg, respectively. Our method is more efficient, easier, and cheaper than conventional clean-up procedures (liquid–liquid extraction)

OCHRATOXIN A RESIDUES IN HUNTED WILD BOAR (SUS SCROFA) FROM TUSCANY

Ochratoxin A (OTA) is a secondary toxic metabolite synthesized by Aspergillus or Penicillium species, which can contaminate various crops. The International Agency for Research on Cancer classified OTA as a group 2B possible human carcinogen. OTA is nephrotoxic, mutagenic, teratogenic and immunosuppressive. OTA can also be present in meat of animals where its presence comes as a result of animal feeding with contaminated grain and feed mixtures. The Italian Ministry of Health Circular No 10, dated 9 June 1999, establishes, as a guideline, a maximum value of 1 μg/kg OTA for swine meat and meat products. The significant increase in the wild boar population has resulted in an increased prevalence of wild boar meat, offal and ready-made products in the food industry. The aim of the present study was to determine OTA concentrations in muscle, kidney and liver of wild boar hunted in Tuscany region. A total of twenty wild boars (male n=11 female n=9) were collected in the Province of Pisa from November 2014 to April 2015, animals have been slaughtered and the carcass weight were determined (from a min. of 14.9 kg and a max. of 72.0 kg). Samples of kidney, liver and muscles from each wild boar were collected and analyzed with an enzymatic digestion clean-up and high-pressure liquid chromatography with fluorescence detection method (1). The highest levels of OTA were found in the kidneys of the twenty wild boar analyzed (0.07- 2.01 μg/kg, mean 0.58±0.63 μg/kg). The levels found in the liver ranged between 0.08- 1.93 μg/kg, (mean 0.53±0.60). The lowest concentrations were found in muscle (0.04- 0.77 μg/kg, mean 0.24±0.24). In eight samples of the tissue samples examined in this study (4 kidney and corresponding 4 liver), the levels of OTA were higher than the guideline level (1 μg/kg) established by the Italian Ministry of Health. The present results are in agreement with a previous study conducted in Calabria in wild boars (2). Swine are particularly sensitive to OTA, kidneys showed the highest accumulation of the latter 101 Società Italiana delle Scienze Veterinarie toxin, followed by liver and muscle tissue, finally the lowest accumulation is represented in adipose tissue. The present results showed the same type of accumulation in wild boar. Traditionally in Tuscany, as in other regions, wild boar meats are used to produce niche products, especially coppa and salami. In agreement with the research of Monaci et al. (3), dried wild boar meat may contribute to overall OTA intake by carry-over effects into processed meats. Monitoring the quality of meat destined for transformation is a priority in order to decrease the possibility of toxin carry-over to humans. The present study confirms that contamination of meat products by OTA represents a potential emerging source of OTA for distinct segments of the Italian population, who are significant consumers of locally-produced wild boar specialties. 1. Luci G., Vanni M., Ferruzzi G., Mani D., Intorre L., Meucci V. 2016. MethodsX 3: 171-177. 2. Bozzo G., Ceci E., Bonerba E., Di Pinto A., Tantillo G., De Giglio E. 2012. Toxins (Basel) 4: 1440-1450. 3. Monaci L., Tantillo G., Palmisano F. 2004. Analytical and Bioanalytical Chemistry 378: 1777- 1782

Serum levels of Ochratoxin A in dogs with chronic kidney disease (CKD): a retrospective study

Ochratoxin A (OTA) is a mycotoxin produced by secondary metabolism of several fungi belonging to the genera Aspergillus and Penicillium. OTA is potentially nephrotoxic, neurotoxic, immunotoxic and carcinogenic in several animal species and in humans. This toxin has been detected in several human food and animal feed. The aim of this study was to determine OTA in blood samples of healthy and affected by chronic kidney disease (CKD) dogs. CKD group showed higher incidence of OTA-positivity than healthy dogs (96% vs. 56%) and a significantly higher median value of OTA plasma concentration (0.008 ng/ml vs. 0.144 ng/ml). No significant correlation was observed between OTA levels and creatinine values in CKD dogs. This is first study regarding OTA detection in plasma samples of healthy and CKD dogs; the presence of this toxin is higher in nephropatic patients but is not yet clear, if it is correlated with progression of the disease

Ecotoxicological properties of ketoprofen and the S(+)‐enantiomer (dexketoprofen): Bioassays in freshwater model species and biomarkers in fish PLHC‐1 cell line

The increased use of non-steroidal anti-inflammatory drugs (NSAIDs) has resulted in their ubiquitous presence in the environment. The toxicological properties of these two widely prescribed NSAIDs, namely - racemic ketoprofen (rac-KP) and its enantiomer S(+)-ketoprofen (dexketoprofen, DKP) were evaluated. Firstly, by acute and chronic toxicity tests using three representative model organisms (Vibrio fischeri, Pseudokirchneriella subcapitata and Ceriodaphnia dubia). Secondly, by evaluating the responses of biotransformation systems and multidrug resistance associated proteins (MRP1/MRP2) using the PLHC-1 fish hepatic cell-line. Toxicity data from both acute and chronic DKP exposure indicated higher sensitivity through inhibition of bioluminescence and algal growth and through increased mortality/immobilization compared to rac-KP exposure. The growth inhibition test showed that rac-KP and DKP exhibited different values for EC50 (240.2 µg/L and 65.6 µg/L, respectively). Furthermore, rac-KP and DKP did not exert cytotoxic effects in PLHC-1 cells, and produced compound-, time- and concentration-specific differential effects on CYP1A and GST levels. For CYP1A, the effects of rac-KP and DKP differed at transcriptional and catalytic level. Exposure to rac-KP and DKP modulated MRP1 and MRP2 mRNA levels and these effects were also dependent on compound, exposure time and concentration of the individual drug. The present study revealed for the first time, the interactions between these NSAIDs and key detoxification systems, and different sensitivity to the racemic mixture compared to its enantiomer. This article is protected by copyright. All rights reserved