The effect of selected environmental Fusarium mycotoxins on the ovaries in the female wild boar (Sus scrofa).

The contamination of agricultural crops with Fusarium mycotoxins poses one of the greatest problems in food production. Wild boars live in specific habitats and are physiologically sensitive to Fusarium mycotoxins, therefore, they are an interesting model for studies investigating the effects of the discussed toxin, in particular under low-dose exposure. The objective of this study was to determine potential effects of Fusarium mycotoxins ingested with naturally contaminated food on reproductive function based on the proliferation and apoptotic indices of ovarian follicles in female wild boars. The experiment was conducted on 40 wild boars inhabiting north-eastern Poland. The effect of seasonal variations in the quantity and quality of ingested food on the concentrations of Fusarium mycotoxins and their metabolites in the blood of wild boars was analyzed. The observed differences in toxin levels were accompanied by changes in proliferation and apoptotic indices. Proliferation processes were most intense in autumn-winter and were least advanced in winter-spring. The intensity of apoptotic processes was inversely correlated with proliferation.

Multi-residue determination of antibiotics in fish by liquid chromatography-tandem mass spectrometry.

A multi-residue method for the determination of 34 antibacterial drugs (three aminoglycosides, nine ß-lactams, nine fluoroquinolones, three macrolides, five sulfonamides, trimethoprim and four tetracyclines) in fish samples by LC-MS/MS was developed. The procedure enables the isolation of residues based on double extraction of the fish sample with m-phosphoric acid and heptafluorobutyric acid as an ion-pair agent and acetonitrile. All compounds were determined at the same time on a C18 column with gradient elution. The method was validated according to the requirements of European Decision 2002/657/EC. Specificity, recovery, repeatability, within-laboratory reproducibility, decision limit CCα and detection capability CCß were calculated. CCα ranged from 55.3 to 1085 µg kg(-1) and CCß ranged from 59.5 to 1141 µg kg(-1). The overall recoveries were from 96% to 111% with respect to the internal standards.

Identification of flunixin glucuronide and depletion of flunixin and its marker residue in bovine milk.

Residues of flunixin [and its marker residue 5-hydroxyflunixin (5OHFLU)] were determined in milk from cows that intravenously received therapeutic doses of the drug. The samples were collected during each milking (every 12 h) for six consecutive days, and concentrations of flunixin and its metabolites were determined by the method with and without enzymatic hydrolysis (beta-glucuronidase). The highest flunixin concentration in milk was observed 12 h after dosing (2.4 ± 1.42 µg/kg, mean ± SD). Flunixin concentrations in the samples determined with enzymatic hydrolysis were significantly higher (P < 0.05), which suggests the transfer of flunixin glucuronide to the milk. Additionally, unambiguous identification of flunixin glucuronide in the bovine milk was performed with linear ion-trap mass spectrometry. The 5OHFLU concentrations analyzed without enzymatic hydrolysis (22.3 ± 16.04 µg/kg) were similar to this obtained with enzymatic hydrolysis. Flunixin and 5OHFLU concentrations dropped below the limits of detection at 48 h after last dosing.

Antimicrobial susceptibility of Brachyspira hyodysenteriae isolated from 21 Polish farms.

Swine dysentery (SD) is a common disease among pigs worldwide, which contributes to major production losses. Antimicrobial susceptibility testing of B. hyodysenteriae, the etiological agent of SD, is mainly performed by the agar dilution method. This method has certain limitations due to difficulties in interpretation of results. The aim of this study was the analysis of antimicrobial susceptibility of Brachyspira hyodysenteriae (B. hyodysenteriae) Polish field isolates by broth microdilution procedure. The study was performed on 21 isolates of B. hyodysenteriae, collected between January 2006 to December 2010 from cases of swine dysentery. VetMIC Brachyspira panels with antimicrobial agents (tiamulin, valnemulin, doxycycline, lincomycin, tylosin and ampicillin) were used for susceptibility testing of B. hyodysenteriae. The minimal inhibitory concentration (MIC) was determined by the broth dilution procedure. The lowest antimicrobial activity was demonstrated for tylosin and lincomycin, with inhibition of bacterial growth using concentrations > 128 microg/ml and 32 microg/ml, respectively. In the case of doxycycline, the MIC values were < or = 2.0 microg/ml. No decreased susceptibility to tiamulin was found among the Polish isolates and MIC values for this antibiotic did not exceed 1.0 microg/ml. The results of the present study confirmed that Polish B. hyodysenteriae isolates were susceptible to the main antibiotics (tiamulin and valnemulin) used in treatment of swine dysentery. Further studies are necessary to evaluate a possible slow decrease in susceptibility to tiamulin and valnemulin of B. hyodysenteriae strains in Poland.

Application of real-time PCR for detection of Lawsonia intracellularis and Brachyspira hyodysenteriae in fecal samples from pigs.

The aim of the study was to develop and validate real-time PCR method for the quantification of Lawsonia intracellularis and Brachyspira hyodysenteriae in porcine feces. Before the optimization process was performed two different extraction methods were compared to select the more efficient one. Based on the results achieved at this stage the boiling procedure was rejected and a commercially available silica-membrane based method was chosen for further analysis. The primers and the Taqman probe for B. hyodysenteriae and L. intracellularis were based on the sequence of NADH oxidase gene and 16S rDNA gene, respectively. The detection limit of the real-time PCR for suspension of feces inoculated with B. hyodysenteriae and L. intracellularis was determined to be 1.5x10(3) CFU/ml and 6.5x10(1) CFU/ml, respectively. The results of this study demonstrate that our real-time PCR is able to detect low number of B. hyodysenteriae and L. intracellularis cells which is satisfying in routine diagnosis of swine dysentery and proliferative enteropathy. Therefore, it is possible to identify both subclinically infected pigs and those representing an acute form of mentioned diseases. In summary, the quantitative real-time PCR is useful for routine diagnosis of L. intracellularis and B. hyodysenteriae. Compared to conventional PCR, the new validated quantification method based on real-time PCR is fast and with reduced risk of laboratory contamination. The novel technique is specific and even more sensitive than the previously used one. Furthermore, the new real-time PCR enables quick detection and quantification of both pathogens in fecal samples, which helps to estimate the health status of a pig herd.

Residues of veterinary medicinal products and coccidiostats in eggs--causes, control and results of surveillance program in Poland.

The use of veterinary medicinal products in food producing animals for a variety of purposes causes that their residues may be presented in edible tissues. As a result, in concern of public health, European Union Countries establish each year monitoring plans and they control the levels of harmful substances in food of animal origin. This paper presents survey of residues of veterinary medicinal products and coccidiostats in eggs for Poland and European Union in years 2007-2010. Despite the decrease in reported non-compliant results for coccidiostats, the numbers were still higher than those for veterinary medicines. The most often determined coccidiostats were: nicarbazin, dinitrocarbanilide, salinomycin and lasalocid, and the most often reported non-compliant results for veterinary medicines were: antimicrobials, enrofloxacin and doxycycline.

Immunohistochemical detection of Lawsonia intracellularis in tissue sections from pigs.

The aim of the present study was to develop an immunohistochemical method (IHC) for detection of Lawsonia intracellularis (L. intracellularis) in formalin-fixed, paraffin embedded sections of intestines from pigs and to implement this method in differential diagnosis of swine diseases with diarrhea in postweaning pigs. The study was conducted on 165 sections of intestines (ileum, caecum and colon) collected from 76 pigs, representing 42 Polish pig farms. The animals included in the analysis suffered from diarrhea, with bloody or grey to brown feces, and were suspected of porcine proliferative enteropathy (PPE). Sections of intestines were analyzed for the presence of L. intracellularis by polymerase chain reaction (PCR) and IHC. Among 165 intestinal samples from pigs with diarrhea, L. intracellularis DNA was detected by PCR in 33 (20.0%) samples. In this group, 30 samples (18.2% of all the samples tested) were also found positive in IHC, while only 3 (1.8%) were IHC-negative. One hundred thirty-two (80.0%) samples were negative in both tests. The PCR- and IHC-positive samples originated from 11 pigs, 4- to 20-week old, from 8 farms. L. intracellularis antigen was visualized by IHC mostly in intestinal crypts and/or in mononuclear cells of the lamina propria). The positive signal in epithelial cells was observed close to the luminal borders, creating typical specifically stained rims around the crypt lumina. The results of the present study further confirm the usefulness of IHC in the detection of L. intracellularis antigen in the intestinal tissues.

Characterization of autonomic nerve markers and lymphocyte subsets in the ileal Peyer's patch of pigs infected experimentally with Brachyspira hyodysenteriae.

The aim of the present study was to investigate potential interrelationships between immune and neural elements of Peyer's patches in normal pigs (n=8) and in pigs infected experimentally with Brachyspira hyodysenteriae and suffering from swine dysentery (n=8). Assessment of tissue concentration of neuropeptides by enzyme linked immunosorbent assay revealed increased levels of galanin (GAL) and substance P (SP) in samples from the infected animals. In contrast, concentrations of vasoactive intestinal polypeptide (VIP) and somatostatin (SOM) were similar in both groups. Immunohistochemistry demonstrated reactivity of nerve fibres with antibodies specific for dopamine ß hydroxylase, vesicular acetylcholine transporter, SOM, GAL, VIP and SP in the interfollicular region and peripheral areas of the Peyer's patch lymphoid follicles. In the dysenteric pigs, the GAL-positive nerve fibres were more numerous and more intensely labelled than those in the normal animals. Flow cytometry revealed a decreased percentage of CD21(+) lymphocytes and lymphocytes expressing T-cell receptor (TCR)-γ, with or without CD8 (TCR-γ(+)CD8(-) and TCR-γ(+)CD8(+)), in the dysenteric pigs as compared with the normal animals. Percentages of other lymphocyte subsets (CD2(+), CD4(+), CD5(+), CD8(+), CD5(-)CD8(+)) were comparable between the groups. Immunohistochemical investigations generally correlated with results obtained by flow cytometry related to lymphocyte subpopulations. Swine dysentery can therefore affect neuroimmunomodulatory processes in the ileal Peyer's patch, in addition to the large intestine. GAL and SP may play a specific role in this neuroimmune cross-talk.

Limits and regulations for mycotoxins in food and feed.

Mycotoxins are natural contaminants whose presence in food- and feedstuffs cannot be completely avoided. Since several mycotoxins have been associated with and implicated in human and animal diseases there is a need to establish maximum levels, guidelines or action levels for them in some kinds of commodities. International and government authorities in many countries have been investing in mycotoxins research and initiating administrative actions for elaboration of legislation and implementing regulatory measures for the control of mycotoxins. Codex Alimentarius Commission is established international legislation on food and feed. In European Union specific limits and regulations for mycotoxins and other contaminants are constructed under the general Codex standards and based on proposal from European Commission. The legal basis for European Commission became available with the framework Council Regulation (EEC) No 315/93. In this paper, legislation regarding maximum levels for certain mycotoxins in food- and feedstuffs in European Community and other countries were reviewed and discussed.

Sample preparation for residue determination of gentamicin and neomycin by liquid chromatography.

The effect of sample preparation on the determination of gentamicin and neomycin residues in animal tissues was investigated. The extract was mixed with an ion-pair reagent and applied to an octadecyl cartridge. The cartridges were washed with buffer followed by water, and analytes were eluted with ion-pair buffer-acetonitrile mixture. The aminoglycosides were derivatized with 9-fluorenylmethyl chloroformate prior to liquid chromatography using a reversed-phase column and fluorescence detection. Under the conditions applied neomycin was fully separated from all the gentamicin compounds. The highest recoveries of gentamicin and neomycin from spiked tissues were obtained using trichloroacetic acid after initial extraction with phosphate-buffered saline. No interfering peaks from endogenous compounds of matrix were noted at the elution position of the analytes. An intra-laboratory validation of the whole procedure was performed. The calibration graphs were linear from 0.1 to 1.0 mg/kg for gentamicin, and from 0.2 to 1.0 mg/kg for neomycin. Limits of detection were 0.05 mg/kg and 0.10 mg/kg for gentamicin and neomycin, respectively. Limits of quantitation for gentamicin and neomycin were 0.1 and 0.20 mg/kg muscle, liver or kidney tissue, respectively. Recoveries of gentamicin spiked at levels of 0.1 mg/kg porcine tissues ranged from 76 to 86%. Recoveries of neomycin spiked at levels of 0.2 mg/kg porcine tissues ranged from 77 to 83%. The validated procedure was used to determine gentamicin concentrations in porcine tissue after dosing with gentamicin at a level of 5 mg/kg body mass.

Effects of the matrix and sample preparation on the determination of fluoroquinolone residues in animal tissues.

The effects of aqueous-organic solvent extraction and enzymic digestion sonication procedure on the isolation of spiked fluoroquinolones from poultry tissue have been studied. The highest recovery from spiked tissue was obtained using a mixture of trichloroacetic acid-acetonitrile (8:2) as extractant and an SDB1 cartridge for clean-up purposes. Validation data are presented for enrofloxacin, ciprofloxacin, sarafloxacin and difloxacin. The spiking procedure (spike contact time, spike solvent and matrix) had a small influence on the recovery of fluroquinolones from poultry muscle or liver. The effects of a different extraction on the determination of incurred enrofloxacin and its metabolite, ciprofloxacin, residues in poultry tissues have been investigated. The extraction procedures investigated--aqueous-organic solvent extraction, enzymic digestion or sonication--all gave similar results for incurred fluoroquinolone concentration in poultry muscle after correlation for spike recovery. The highest results were obtained in poultry liver when enzymic digestion has been used.

Postimplantation whole embryo culture assay for hamsters: an alternative to rat and mouse.

Postimplantation whole embryo culture (WEC) assay for rats and mice has been well established and introduced to many laboratories. Recently WEC technique for rabbits has been developed; however, information on culture of other species is very limited. Knowing the usefulness of hamsters in classical embryotoxicology, we reasoned that hamster WEC could be an alternative model for the most frequently used rat and mouse WEC. Previously we have optimized culture conditions for postimplantation hamster embryos. The aim of this study was to test the susceptibility of hamster embryos cultured in vitro to embryotoxic compounds and to compare our results with those reported by others on rat or mouse embryo culture. For that purpose we choose three known embryotoxic compounds--valproic acid, cadmium chloride, and diethylstilbestrol--and tested them using a postimplantation hamster whole embryo culture assay. Hamster embryos were cultured from 7.5 days gestation for 24 h in a medium consisting of 35% hamster serum and 65% synthetic culture medium (Iscove's or McCoy 5A). At the end of the culture period, the embryos were examined morphologically, measured with the aid of a computer image analysis system, and total protein content was assessed. All three compounds exhibited dose-related embryotoxic and teratogenic effects in hamster embryos. The malformations observed were similar to those reported on rat and mouse embryos. Comparison of the results with data reported by other authors indicates that hamster embryos cultured in vitro might be more susceptible to embryotoxic stimuli than rat and mouse embryos.

Validation study for the determination of tetracycline residues in animal tissues.

An interlaboratory study of the liquid chromatographic (LC) determination of tetracyclines--oxytetracycline (OTC), tetracycline (TTC), and chlortetracycline (CTC)--in animal tissues was conducted. Isolation was performed with oxalic buffer followed by dechelation and deproteination with oxalic acid-acetonitrile. The extract was cleaned with a styrene-divinylbenzene cartridge. LC analysis was performed with a PLRP-S column and 0.01 M oxalic acid-acetonitrile (75 + 25, v/v) as mobile phase. Participants analyzed 2 control and 10 fortified porcine muscle and kidney samples. Additionally, porcine muscle samples containing incurred residues of tetracyclines were analyzed. Mean recoveries of fortified residues from porcine tissue ranged from 76.00 to 86.89%. Repeatabilities varied from 2.05% for OTC to 3.61% for TTC for muscle samples and from 6.75% for CTC to 8.74% for OTC for kidney samples. Reproducibilities ranged from 2.05 to 4.30% for muscle samples and from 15.77 to 18.81% for kidney samples.

Determination of fluoroquinolone residues in animal tissues by liquid chromatography.

A simple liquid chromatographic (LC) method was developed for the determination of fluoroquinolones (ciprofloxacin, difloxacin, enrofloxacin and sarafloxacin) in animal tissues. Isolation of fluoroquinolones from biological matrices was performed with 5% trichloroacetic acid-acetonitrile (7:3) solution. For clean-up, solid-phase extraction with an SDBI (styrene-divinylobenzene) cartridge was used. LC analyses were performed with analytical column (LiChrospher 100 RP-8 5 microns) and mobile phase (0.025 M o-phosphoric acid-acetonitrile 70:30, v/v) in ion-pair mode. The whole procedure was validated in intra- and inter-assay reproducibility and accuracy determination by simultaneously assaying of muscle, liver and kidney samples supplemented with fluoroquinolones at the level of 30 and 60 ng/g, respectively. The statistical evaluation demonstrates high absolute recovery (> 80%) and low coefficient of variation (< 10%) for all analysed samples. The detection limits for fluoroquinolones were 5 ng/g in muscle, liver and kidney samples.

Determination of tetracycline residues in animal tissues by liquid chromatography.

A simple liquid chromatographic (LC) method was developed for the determination of tetracyclines (oxy-tetracycline, tetracycline and chlortetracycline) in animal tissues. Isolation of tetracyclines from biological matrices was performed with oxalic buffer followed by dechelation and deproteinization with oxalic acid-acetonitrile solution. For clean-up solid phase extraction with a SDBI (styrene-divinylbenzene) cartridge was used. LC analysis was performed on a polymeric analytical column (PLRP-S 5 microns, 150 x 4.6 mm) and using an oxalic acid mobile phase (0.01 M oxalic acid - acetonitrile 75:25, v/v). The whole procedure was validated for intra- and inter-assay reproducibility determination by assaying muscle, liver and kidney samples supplemented with tetracyclines at the level of 50, 100 and 200 ng/g, respectively. The statistical evaluation demonstrates high absolute recovery (> 80%) and low coefficient of variation (< 10%) for all analysed samples. The detection limits for tetracyclines were 10-15 ng/g in muscle, and 20-25 ng/g in liver and kidney samples, depending on the analyte.

Tissue concentration of dimetridazole in laying hens.

After laying hens had been dosed orally with dimetridazole (DMZ) for 3 days (50 and 250 mg/kg body weight (b.w.)) or intramuscularly, also for 3 days (50 mg/kg b.w.), the residues were determined in serum, liver, breast and thigh muscle by liquid chromatography. The limit of determination was 0.01 micrograms/g. The maximum concentration of DMZ was found at 1 h following application. After oral doses (50 and 250 mg/kg b.w.) no residues were found in muscle (breast and thigh) at 48 and 72 h, respectively. After intramuscular injection, residues in thigh muscle were below 0.01 micrograms/g at 72 h but breast muscle (injection site) still had concentrations above this level. Bioavailability (F > 80%) and some pharmacokinetic parameters were determined. The elimination half-lives from serum were 2.56h and 2.69h, after both oral doses, respectively, and 2.88 h after intramuscular application.

[Aromatic chlorinated hydrocarbons in in food of animal origin]. / Chlorowane weglowodory aromatyczne w zywnosci zwierzecego pochodzenia.

During 1990-1994 samples of fat tissues from about 18000 animals (swine, cattle, horses, rabbits, poultry and game animals), about 300 pooled milk samples and over 450 eggs were taken from the all 49 districts of Poland. Levels of organochlorine pesticides and polychlorinated biphenyls (PCB) were determined by capillary gas chromatography. The highest mean concentrations of DDT were found in fat of village hens (0.56 mg/kg) and fat of intensive rearing hens (0.2 mg/kg). In swine, cattle and game animals these levels were 0.1 mg/kg and below. Other organochlorine pesticides (mainly HCH isomers and HCB) were found rather in low levels and only in some percentage of tested samples. Results of our 20 year study indicate that DDT levels in animal fat has decreased 20 times during that time. The highest mean concentrations of PCB were found in horses (0.08 mg/kg of fat) and from 0.02 mg/kg to 0.05 mg/kg in fat of other species, milk and eggs.

Residues of dimetridazole in eggs after treatment of laying hens.

Laying hens were dosed orally with dimetridazole (DMZ) (50 and 250 mg/kg) for 3 days or intramuscularly (50 mg/kg), also for 3 days, and the residues were determined by liquid chromatography in albumen and yolk. The sensitivity of the whole procedure was 2 ng/g. The drug was excreted preferentially into the yolk (about 57% of the total) and the elimination period lasted for 4-6 days after treatment.

[Residues of chlorinated hydrocarbons in cheeses]. / Pozostalosci chlorowanych weglowodorów aromatycznych w serach.

In 1995 samples of curdy cheeses without fermentation (n = 112) and rennet manner fermented cheeses (n = 124) were taken in all districts of Poland. Levels of organochlorine pesticides and polychlorinated biphenyls (PCB) were determined by capillary gas chromatography. In almost all cheese samples DDT and PCB residues were found. The mean concentration of sigma-DDT in fat of curdy cheeses was 0.058 mg/kg (from 0.010 mg/kg to 0.181 mg/kg) and PCB 0.013 mg/kg (from traces to 0.048 mg/kg). Other organochlorine pesticides (mainly HCB and HCH isomers) were measured in low levels and only in some samples. Residues of chlorinated hydrocarbons in fermented cheeses were on the same levels as in curdy cheeses. The mean concentration of sigma-DDT in fat of fermented cheeses was 0.061 mg/kg and PCB 0.012 mg/kg. The maximum residue limit of pesticides was not exceeded in any cheese samples.