Effects of broccoli extract and various essential oils on intestinal and faecal microflora and on xenobiotic enzymes and the antioxidant system of piglets

Objective: Since the ban of antibiotics as growth promoting feed additives in the EU in 2006 research in alternatives has gained importance. Phytogenic feed additives represent a heterogenous class of different plant derived substances that are discussed to improve the health of farm animals by direct and indirect antioxidant effects and by influencing microbial eubiosis in the gastrointestinal tract. Consequently our study aimed to investigate the influence of broccoli extract and the essential oils of tur- meric, oregano, thyme and rosemary, as selected individual additives, on intestinal and faecal microflora, on xenobiotic enzymes, and on the antioxidant system of piglets. Methods: 48 four weeks old male weaned piglets were assigned to 6 groups of 8. The piglets were housed individually in stainless steel pens with slatted floor. The control group (Con) was fed a diet without an additive for 4 weeks. The diet of group BE contained 0.15 g/kg sulforaphane in form of a broccoli extract. 535, 282, 373 and 476 mg/kg of the essential oils of turmeric (Cuo), oregano (Oo), thyme (To) and rosemary (Ro) were added to the diets of the remaining 4 groups to stan-dardise supplementation to 150 mg/kg of the oils’ key terpene compounds ar-turmerone, carvacrol, thymol and 1,8-cineole. The composition of bacterial microflora was examined by cultivating samples of jejeunal and colonic mucosa and of faeces under specific conditions. The mRNA expression of xenobiotic and antioxidant enzymes was determined by reversing transcrip- tase real time detection PCR (RT-PCR). Total antioxidant status was assayed using the Trolox Equivalent Antioxidant Capacity (TEAC), and lipid peroxidation was determined by measuring thiobarbioturic acid reactive substances (TBA- RS). Results: Compared to Con piglets all additives positively influenced weight gain and feed conversion in week 1. Over the whole trial period no significant differences in performance parameters existed between the experimental groups. Compared to group Con performance of Ro piglets was, however, slightly impaired. Com- pared to Con piglets Cuo, Oo and To increased the ratio of Lactobacilli:E. coli attached to the jejunal mucosa, whereas BE and Ro impaired this ratio slightly. In contrast in colonic mucosa Ro improved Lactobacilli:E. coli ratio. In faecal samples an improvement of Lactobacilli:E. coli ratio could be analysed for To and Ro. Ro was the only additive that reduced the incidence rate of piglets tested positive for enterotoxic E. coli (ETEC). All additives significantly increased jejunal TEAC and reduced TBA-RS. In the liver BE, Cuo, Oo and To increased TEAC in tendency and Ro significantly. Liver TBA-RS were slightly reduced by all additives compared to Con piglets. Whereas the influence of BE, To and Ro on jejunal TEAC mainly was derived from the induction of xenobiotic and antioxidant enzymes (indirect antioxidant effects), Cuo and Oo influenced TEAC by direct antioxidant effects. Discussion and Conclusions: Our results have shown: That within the labiatae oils Oo and To have the potential to improve performance slightly. That phytogenic substances have a small but not sig- nificant influence on intestinal microflora. That phytogenic feed additives up-regulate the anti- oxidant system of piglets either by direct or by indirect antioxidant effects and that they may thereby improve health status. That within the labiatae oils Oo has a high direct antioxidant potential whereas Ro potently induces xenobiotic and antioxidant enzymes. That broccoli extract is an attractive new phytogenic additive, improving antioxidant status by indirect antioxidant effects. That defined combinations of selected phytogenic substances may produce additive effects. That health promoting effects of phytogenic additives in the future should be studied systematically under the challenge with pathogenic microorganisms or food derived to-xins.

Prion Protein Binds to Aldolase A Produced by Bovine Intestinal M Cells

Microfold (M) cells are a kind of intestinal epithelial cell in the follicle-associated epithelium (FAE) of Peyer’s patches. They can transport antigens and microorganisms to lymphoid tissues. Bovine spongiform encephalopathy (BSE) is a fatal neurodegenerative disorder in cattle. It is linked to variant Creutzfeldt-Jakob disease in humans. Although it is thought that M cells transport the BSE agent, the exact mechanism by which it crosses the intestinal barrier is not clear. We have bovine intestinal epithelial cell line (BIE cells), which can differentiate into the M cell type in vitro after stimulation, and which is able to transport the BSE agent. We show here that M cells are able to incorporate large numbers of PrP coated magnetic particles into intracellular vesicles, which we collected. The results of 2-DE show a specific protein associated with the PrP-coated particles, compared with non-coated particles. This protein was identified as aldolase A, a glycolytic pathway enzyme, using LC-MS/MS analysis. Aldolase A was synthesized and secreted by BIE cells, and increased during M cell differentiation. In the villi of the bovine intestine, aldolase A was detected on the surface of the epithelium and in the mucus droplet of goblet cells. In the FAE of bovine jejunal and ileal Peyer’s patches, aldolase A was localized on the surface and the apical part of the M cells. The binding of rbPrP to aldolase A was clearly detected and inhibited by pre-treatment of anti-aldolase A antibody. Aldolase A was co-stained with incorporated PrPSc in M-BIE cells. These results suggest that bovine M cells and goblet cells synthesize aldolase A, and that aldolase A may have the ability to bind PrP and associate with PrP in cellular vesicles. Therefore, aldolase A-positive M cells may play a key role in the invasion of BSE into the body.

Determination of thyreostats in bovine urine using ultra-high performance liquid chromatography-tandem mass spectrometry

Five thyreostats(TSs),namely tapazole,thiouracil,methylthiouracil,propylthiouracil,and phenylthiouracil,were determined in bovine urine using ultra-high performance liquid chromatography-tandem mass spectrometry(UHPLC-MS/MS)in positive electrospray ionization mode.Extraction and clean-up were achieved using a ChemElut cartridge with tert-butyl methyl ether,without a derivatization step.Separation was achieved on an Acquity UPLC SS T3 column.The mobile phase was acetonitrile and water containing 0.2%(v/v)formic acid.The mass spectrometer was operated in multiple reaction monitoring mode.Urine samples were spiked with TS solution at levels corresponding to 5,10,15,and 20μg/L.The accuracy(internal standard corrected)ranged from 92%to 107%,with a repeatability precision(relative standard deviation,RSD)less than 15%for all five analytes.The RSDs within-laboratory reproducibility was less than 26%.The decision limits(CCα)and detection capabilities(CCβ)were obtained from a calibration curve and were in the ranges of 3.1-6.1μg/L and 4.0-7.4μg/L,respectively.The CCαand CCβvalues were below the recommended concentration,which was set at 10μg/L.The results show that the described method is suitable for the direct detection of TSs in bovine urine.This method can also be used to determine TSs in porcine urine.

Diversity of livestock associated methicillin-resistant Staphylococcus aureus

Objective: To evaluate the diversity and molecular characteristics of livestock associated methicillin-resistant Staphylococcus aureus in livestock animals, food of animal origin and the environment in the Czech Republic.Methods: After having been primarily enriched in buffered peptone water, the samples were cultured on Baird–Parker agar. Presumptive colonies were sub-cultured to blood agar and assessed morphologically. Furthermore, presumptive Staphylococcus aureus colonies were confirmed by MALDI-TOF. Multiplex PCR, spa-typing, and MLST have been used to characterize the strains. Each mec A-positive Staphylococcus aureus isolates were examined against 14 different antimicrobials by using disk diffusion method.Results: In this study, 13 different spa-types belonging to five sequence types(ST) were detected. Ninety four percent of tested strains belonged to CC/ST398 for which t011,t034, t2123 and t2346 were the vast major spa-types. In addition, non-ST398 clones such as CC1(t127), ST5(t3598), ST8(t064) and ST361(t315) were detected, which are known as human associated clones.Conclusions: The diversity of livestock associated methicillin-resistant Staphylococcus aureus has grown, and detecting lineages of human origin in animals and vice-versa becomes more common. Thus, livestock animal and its products will be a potential for the evolvement of methicillin-resistant Staphylococcus aureus in human population.Monitoring of pigs as well as other food-producing animal species and their products is therefore recommended.

Chemical Quality Indexes of Mullet (<i>Mugil platanus</i>) Stored on Ice

The present study was conducted aiming at establishing chemical quality parameters to assess ice stored mullet (0℃ ± 1℃) through the evaluation of nucleotide (adenosine monophosphate [AMP], inosine [HxR] and hypoxanthine [Hx]) degradation, biogenic amine (histamine [HI], putrescine [PU], cadaverine [CA] and tyramine [TI]) quantification and mesophilic and psychrotrophic bacteria count monitoring. The microbial load of 7 log CFU·g–1 established as maximum acceptable limit was attained after the 20th day of ice storage. IMP concentration declined during the storage period to levels below the detection limit. HxR content increased only up to time T3 and then declined. Hx level increased during all the storage period. CA and HI content increase was not observed, on the other hand, PU and TI contents significantly increased (p < 0.05) at time T5. We concluded that IMP and Hx concentrations can be adequate parameters to assess mullet quality under the study conditions. HxR proved to be adequate to evaluate the freshness of mullet in the first days of storage while the amines, PU and TI, can be used to assess loss of quality. Mullet obtained in conditions similar to those of the present study and maintained at 0℃ ± 1℃ can be consumed up to the 20th storage day.

Evaluation of Antibodies Induced by the Injection of Single Capsid Protein or Purified Virus Particle of Coxsackievirus B3 in Mice

Four capsid proteins (VP1, VP2, VP3, and VP4) of coxsackievirus B3 (CVB3) were expressed as recombinant proteins in an Escherichia coli expression system and used as antigens for subunit vaccines against CVB3 in ICR mice. Antigens were expressed as thioredoxin-histidine (TrxHis)-tagged protein and purified before immunization. Although all VPs other than VP4 induced anti-CVB3 specific antibodies in mice (detected by ELISA and western blotting), they did not neutralize the infectious CVB3 in a virus neutralization assay. Meanwhile, 2 virus strains were purified from CVB3 virus stock on the basis of their plaque size on HeLa cells. ICR mice were infected with the 2 purified virus strains (S-strain and L-strain) and unpurified virus stock (wild type) to analyze the difference in antibody responses against infections of purified and unpurified virus strains. The reactivity of antisera against each virus strain was tested by ELISA, and the results showed that the inoculation of purified virus strain induced a strong antibody response against the inoculated strain. As a result, the antibody response against wild-type and other virus strains was suppressed. These results suggest using unpurified virus stock as an antigen is advantageous for inducing a broad antibody response in inoculated animals.

Phenotypic Characterization Murine Sarcoma TG-180 Immunophenotypical Characterization Murine Sarcoma TG-180

The sarcoma is the generic nomenclature for neoplasm of mesodermal cells, which express in man and animals. Silent growth requires early diagnosis technique for identifying their proteins. The experimental model in vivo murine sarcoma 180-TG (TG-180), is widely used in research to provide the stimuli of infectious and neoplastic antigens. In this case, the technique of immunohisto-chemistry helps identify the expressions of tumor cell variants. The objective of the research was to characterize immunoexpression murine sarcoma TG 180, by immunohistochemistry, antibodies AE1/AE3, vimentin, CD3, CD 45, CD79α and S100A4. For this, murine sarcoma TG-180, was implanted subcutaneously in 20 mice “Swiss”, male, 30 days old, 28 g for 10 days. Samples were taken and subjected to immunohistochemistry, via use of HistoMouse-MA&trade;? kit. There was specifically labeled S100A4 and vimentin antibodies, indicative of poorly differentiated neoplasms fibroblasts. In fact, the model is established by identifying the origin of the cell, once identified, chemotherapeutic tests can also be performed. Neoplasia like these, when installed in man and animals, depending on the degree of aggressiveness requires treatment protocol varied between surgery and chemotherapy or combination of treatments.

High Amounts of Halogenated Natural Products in Sperm Whales(Physeter macrocephalus)from Two Italian Regions in the Mediterranean Sea

Halogenated natural products(HNPs)are considered to be emerging contaminants whose environmental distribution and fate are only incompletely known.Therefore,several persistent and bioaccumulative HNP groups,together with manmade polychlorinated biphenyls(PCBs)and polybrominated diphenyl ethers(PBDEs),were quantified in the blubber of nine sperm whales(Physeter macrocephalus)stranded on the coast of the Mediterranean Sea in Italy.The naturally occurring polybrominated hexahydroxanthene derivatives(PBHDs;sum of TetraBHD and TriBHD)were the most prominent substance class with up to 77,000 ng/g blubber.The mean PBHD content(35,800 ng/g blubber)even exceeded the one of PCBs(28,400 ng/g blubber),although the region is known to be highly contaminated with manmade contaminants.Based on mean values,Q1∼PBDEs>MeO-BDEs∼2,2′-diMeO-BB 80 and several other HNPs followed with decreasing amounts.All blubber samples contained an abundant compound whose molecular formula(C_(16)H_(19)Br_(3)O_(2))was verified using high-resolution mass spectrometry.The only plausible matching isomer was(2S,4′S,9R,9′S)-2,7-dibromo-4′-bromomethyl-1,1-dimethyl-2,3,4,4′,9,9′-9,9′-hexahydro-1H-xanthen-9-ol(OH-TriBHD),a hydroxylated secondary metabolite previously detected together with TriBHD and TetraBHD in a sponge known to be a natural producer of PBHDs.The estimated mean amount of the presumed OH-TriBHD was 3000 ng/g blubber,which is unexpectedly high for hydroxylated compounds in the lipids of marine mammals.