In-house validation method for quantification of formaldehyde in silage and feedingstuffs with the use HPLC-DAD technique.

An HPLC-DAD method was developed for the determination of formaldehyde in animal feed and silage. The method is based on the determination of the product of chemical reaction between formaldehyde and 2,4-dinitrophenylhydrazine. A 3 g of feed or silage were extracted with Milli-Q water with phosphoric acid and next formaldehyde was derivative with the use 2,4-dinitrophenyl- hydrazine in acetronitrile solution. The extract was purified with 0.45 µm syringe filters and separeted on Zorbax Eclipse XDB C18 column and detection was carried out at 360 nm. Formal- dehyde was eluted with a mobile phase consisting of acetonitrile/water in isocratic elution. This method provided average recoveries of 90.6% to 102.2%, with CVs of 2.6% to 6.4% for feed and from 91.3% to 108.7% with CVs of 1.1% to 4.1% for silage in the ranged of 50 to 1000 mg/kg feeds and silage. The LOD and LOQ for formaldehyde in feed and silage ranged from 1.6 to 2.6 and 2.7 to 5.7 mg/kg, respectively. The methodology was applied for the analysis of feed and silage samples collected from poultry, pigs and cows farms.

Development and Validation of an Analytical Method for Determination of Ergot Alkaloids in Animal Feedingstuffs with High Performance Liquid Chromatography-fluorescence Detection.

A high performance liquid chromatography combined with fluorescence detection (HPLC-FLD) method was developed for determination of five ergot alkaloids (EA): ergometrine, ergotamine, ergocornine, ergocrypine and ergocristine in animal feedingstuffs. The method was based on the application of QuEChERS salts for extraction and modified QuEChERS dispersive SPE for the cleanup step. Alkaloids separation was performed on a C18, 250 mm x 4.6 mm, 5 µm column with the mobile phase containing ammonium carbonate and acetonitrile. The excitation and emission wavelengths were 330 and 420 nm respectively. The method was validated according to the Commission Decision 2002/657/EC and all parameters are in agreement with the requirements of the Decision. Linearity was determined for the concentration range of 25-400 µg/kg. The coefficient of determination (R2) for all curves was from 0.985 to 0.996. The limit of detection (LOD) was in the range 3.23 to 6.53 µg/kg and the limit of quantification (LOQ) from 11.78 to 13.06 µg/kg. The decision limit (CCα) ranged from 29.56 to 43.08 µg/kg and detection capability (CCß) from 40.65 to 51.01 µg/kg. The highest coefficient of variation (CV) for repeatability was 14.3% and for reproducibility 15.4%.

Molecular diversity of Clostridium botulinum and phenotypically similar strains.

This study was undertaken to examine phenotypic and genetic features of strains preliminary classified as Clostridium botulinum species. The phenotypic characteristics were assessed with different culture media and biochemical tests. The genetic characterization included detection of botulinum toxin genes by PCR and macrorestriction analysis with SmaI, XhoI and SacII by PFGE (Pulsed-field Gel Electrophoresis). Despite similar biochemical properties of all analysed strains, only 47% of them contained genes determining toxicity specific to C. botulinum species. The most valuable differentiation of C. botulinum and C. botulinum-like strains was obtained after SmaI digestion. The highest affinity was observed among C. botulinum type B profiles which was even up to 100%. It was found 100% of affinity between C. botulinum and C. botulinum-like strains, however, the similarity among C. botulinum and C. botulinum-like was generally lower than 80%.

Screening method for the determination of tetracyclines and fluoroquinolones in animal drinking water by liquid chromatography with diode array detector.

A liquid chromatography - diode array detector (HPLC-DAD) procedure has been developed for the determination of oxytetracycline (OTC), tetracycline (TC), chlorotetracycline (CTC), doxycycline (DC), enrofloxacin (ENR), ciprofloxacin (CIP), sarafloxacin (SAR) and flumequine (FLU) residues in animal drinking water. This method was applied to animal drinking water. Solid-phase extraction (SPE) clean-up on an Oasis HLB cartridge allowed an extract suitable for liquid chromatographic analysis to be obtained. Chromatographic separation was carried out on a C18 analytical column, using gradient elution with 0.1% trifluoroacetic acid - acetonitrile - methanol at 30°C. The flow-rate was 0.7 mL/min and the eluate was analysed at 330 nm. The whole procedure was evaluated according to the requirements of the Commission Decision 2002/657/EC, determining specificity, decision limit (CCα), detection capacity (CCß), limit of detection (LOD), limit of quantification (LOQ), precision and accuracy during validation of the method. The recoveries of TCs and FQs from spiked samples at the levels of 10, 100 and 1000 µg/L were higher than 82%. The developed method based on HPLC-DAD has been applied for the determination of four tetracyclines and four fluoroquinolones in animal drinking water samples.

Methods and difficulties in detection of Clostridium botulinum and its toxins.

The aim of this work was to present selected data regarding traditional and modern methods for C. botulinum and its toxins detection. In this article, methods based on culturing techniques, mouse bioassay, immunological techniques, chromatography and PCR, PFGE, RFLP, AFLP are described. The mentioned techniques were evaluated considering their usefulness in the samples examination, genotyping of strains and the diagnostics of botulism.

Assessing the possibility of genetically modified DNA transfer from GM feed to broiler, laying hen, pig and calf tissues.

The aim of this study was to assess the possibility of genetically modified DNA transfer from feed containing RR soybean or/and MON810 maize to animal tissues, gut bacterial flora, food of animal origin, and the fate of GM DNA in the animal digestive tract. The experiment was carried out on broilers, laying hens, pigs and calves. All animals were divided into four groups: I--control group (non-modified feed), II--GM soybean group (non-modified maize, RR soybean), III--GM maize group (MON810 maize, non-modified soybean), and IV--GM maize and soybean group (MON810 maize, RR soybean). Samples of blood, organs, tissues, digesta from the gastrointestinal tract, and eggs were analysed for the presence of plant species specific genes, and transgenic sequences of CaMV 35S promoter and NOS terminator. PCR amplifications of these GM sequences were conducted to investigate the GM DNA transfer from feed to animal tissues and bacterial gut flora. In none of the analysed samples of blood, organs, tissues, eggs, excreta and bacterial DNA were plant reference genes or GM DNA found. A GM crop diet did not affect bacterial gut flora as regards diversity of bacteria species, quantity of particular bacteria species in the animal gut, or incorporation of transgenic DNA to the bacteria genome. It can be concluded that MON810 maize and RR soybean used for animal feeding are substantially equivalent to their conventional counterparts. Genetically modified DNA from MON810 maize and RR soybean is digested in the same way as plant DNA, with no probability of its transfer to animal tissues or gut bacterial flora.

Detection and identification of banned processed animal protein in feedingstuffs by microscopic and PCR methods.

The aim of the study was to present the results of comparative evaluation of the usefulness of PCR and microscopic methods for the detection of Processed Animal Protein (PAP) in feedingstuffs. In the validation study, the limit of the detection for PCR was determined on 0.05% for beef, 0.1% for pork and 0.2% for poultry meat and bone meal (MBM). Among 62 doubtful samples of feedingstuffs examined by microscopic method 41 (66.13%) were found as positive. Based on the results obtained with the use of the microscopic and PCR methods it is possible to state that the molecular biology methods can, at present, be used as a supplementary method in PAP detection.

Enhanced phenotypic and functional maturation of monocyte-derived dendritic cells from patients with active Crohn's disease and ulcerative colitis.

Disturbed immunoregulation and an inappropriate immune response to gut microflora is assumed to be involved in the pathogenesis of inflammatory bowel disease (IBD). Physiologically dendritic cells (DCs) as the professional antigen presenting cells play a crucial role in the control of intestinal inflammation and immune tolerance. In order to evaluate their role in the IBD we analyzed the phenotypic and functional properties of monocyte-derived DCs (MoDCs) generated from UC and CD patients following stimulation with TNF-α, lipopolisaccharide E. coli or hydrocortisone. Thirty seven patients with moderate to severe inflammation (19 UC, 18 CD) were recruited to the study. Monocyte-derived dendritic cell immunophenotypes and their endocytic ability were analysed by flow cytometry and confocal microscopy, IL-6, IL-10, IL-12 and IL-23 secretion were investigated by ELISA. Both unstimulated and stimulated MoDCs generated from IBD patients had more mature phenotype and secreted elevated concentrations of proinflammatory cytokines as compared to a control group. The addition of LPS E. coli to culture media was associated with enhanced dendritic cell activation and maturation as compared to DCs stimulated only with TNF-α. This may suggest altered dendritic cell interactions with intestinal microflora in inflammatory bowel disease. Hydrocortisone decreases the numbers of mature dendritic cells and the proinflammatory cytokine concentrations in all cell culture types that may explain the efficacy of steroid therapy in inflammatory bowel disease.

Is melatonin involved in the irritable bowel syndrome?

There is a substantial evidence that large quantities of melatonin are produced in gastrointestinal tract, however, is still unclear which is the role of melatonin in digestive system in human physiology and pathophysiology. In the present study we investigated urinary excretion of a main melatonin metabolite, 6-sulphatoxymelatonin, in patients with irritable bowel syndrome (IBS). The investigation was carried out in 67 persons, both sexes, aged 20-45 years old who according to Rome III Criteria were diagnosed as sufferers of constipation (C-IBS, n=21 persons) or diarrhoea (D-IBS, n=24 persons) form of irritable bowel syndrome and as healthy subjects (K, n=22), matched for control. Samples were obtained from the collected diurnal urine. The concentration of 6-sulphatoxymelatonin (6-SMLT) was measured with ELISA method, creatinine (crea) was automatically analyzed with biochemical analyzer and 6-SMLT/crea calculated. There were statistically significant differences between groups: the 6-SMLT/crea level was lower in C-IBS (103.86+/- 82.83 ng/mg) and D-IBS (112.72+/-85.29 ng/mg) groups compared to K group (202.7+/-89.28 ng/mg), respectively, p=0.002, p=0.003. There were no differences between C-IBS and D-IBS groups, however, there were observed differences between men and women with C-IBS. The 6-SMLT/crea. level was higher in women with C-IBS (139.31+/-96.45) compared to men with C-IBS (35.51+/-41.05) (p=0.04). These results suggest that different melatonin secretion and metabolism may be involved in the pathogenesis of irritable bowel syndrome.

[Degree of nicotine dependence in the Gastroenterology Ward of Clinical Hospital number 4 in Lublin]. / Stopien uzaleznienia od nikotyny pacjentów hospitalizowanych w Oddziale Gastroenterologii SPSK 4 w Lublinie.

It is common knowledge that smoking badly influences people's health. Amount of toxic chemical substances delivered to the human organism is directly proportional to the number of smoked cigarettes. Tobacco smoking increases the number of ill people and accelerates death. The problem of nicotine addiction has both social and medical aspects. The aim of the work was to assess the tobacco smoking among patients hospitalized in the Gastroenterology Clinic in SPSK 4 in Lublin. We found that smoking prevalence in the investigated group considerably exceeds the average values for Polish population.

Current issues connected with usage of genetically modified crops in production of feed and livestock feeding.

Progress, which is brought by new advances in modern molecular biology, allowed interference in the genome of live organisms and gene manipulation. Introducing new genes to the recipient organism enables to give them new features, absent before. Continuous increase in the area of the biotech crops triggers continuous discussion about safety of genetically modified (GM) crops, including food and feed derived from them. Important issue connected with cultivation of genetically modified crops is a horizontal gene transfer and a bacterial antibiotic resistance. Discussion about safety of GM crops concerns also food allergies caused by eating genetically modified food. The problem of genetic modifications of GM crops used for livestock feeding is widely discussed, taking into account Polish feed law.

Microbiological quality of petfood in Poland.

From epidemiological and safety point of view, petfoods produced from edible and non-edible animal by-products should be safe for slaughter animals, owners of these pets and environment. Domestic animals are recognized as highly important reservoir of pathogenic microorganisms for man. The close contact of people and especially children with pets may constitute a serious hazard for their health. Taking these aspects into account, the study was undertaken in which 2271 dried and 18 canned petfood samples were examined. Moreover, 14, 90, 23 and 22 samples of petfoods were examined for presence or number of Clostridium sp., Enterobacteriaceae, total plate count, and yeast and moulds, respectively. Salmonella was isolated from 22 (1%) out of 2271 dried petfood samples examined. Number of Enterobacteriaceae was higher than 300 cfu/g, i.e. the allowed maximum level was found in 9 (10%) samples examined. The rest of microbiological quality parameters did not exceed the allowed values.

National programme of official control of animal feedingstuffs under surveillance of veterinary inspection in Poland.

Poland as a new EC country is obliged in agree with Directive 95/53/EC to drawn up one, coherent and coordinated national programme of official inspection, irrespective of the organisational structure, and the number of inspection authorities in a country. Taking these facts into account the author has worked out the Polish National Programme of an official control conducted by the Veterinary Inspection in the field of animal feedingstuffs sector in Poland. The objective of the programme is to lay down the substantive scope of official inspections and laboratory control testing of animal feedingstuffs, appropriate for the surveillance exercised by the Veterinary Inspection. The programme is under implementation process which has begun in 2004.