Intraepithelial but not lamina propria lymphocytes in the porcine gut are affected by dexamethasone treatment

It is well established that glucocorticoids are key regulators of the immune system and act as immunosuppressive agents in high concentrations. In the pig, effects on the gut immune system and trafficking of lymphocytes between tissues and blood plasma were not investigated so far. Twelve pigs of 70 kg were fed 0.4 mg portions of dexamethasone (Dexa) twice daily for 9 days or remained untreated (controls) and were sacrificed for tissue collection at the end of Dexa treatment. Another six pigs with jugular vein catheters were left untreated for 7 days (control period) and then received Dexa for 9 days. Blood was drawn twice during the control period and at days 3, 6 and 9 of the Dexa period for characterization of peripheral blood leukocytes. Cells were obtained from thymus, mesenteric lymph nodes, jejunal mucosa and Peyer's patches. Lymphoid cells from gut tissue were isolated from two fractions: the EDTA-fraction, containing the intraepithelial lymphocytes (IEL), and the Collagenase-fraction, containing the lamina propria lymphocytes (LPL). In all samples, cell counts and phenotypic characterization of cells by flow cytometry (FCM) were performed. In thymus, Dexa led to a more than 90% reduction of the absolute cell number, which was mainly found in the CD4+CD8+ subpopulation. Dexa effects on lymphocytes from mesenteric lymph nodes were less severe (50%) and led mainly to a decrease (71%) of B-lymphocytes. The number of lymphocytes in the EDTA-fraction (IEL) of the jejunal mucosa decreased significantly by 56% in the Dexa-treated animals compared to the controls, whereas the number of lymphocytes in the Collagenase-fraction (LPL) decreased only moderately. In the Peyer's patches, a decreasing tendency in the number of lymphocytes in the EDTA-fraction was observed which, however, was not significant. In blood, monocytes and granulocytes were significantly increased in an order of 60%. The data show that supraphysiological amounts of Dexa remarkably reduce cell numbers in thymus and also in the intraepithelial compartment of the jejunal mucosa and ileal Peyer's patches. In blood, a notable homeostasis was observed for several leukocyte populations whereas both monocytes and granulocytes increased.     

Chloramphenicol resistance plasmids in Staphylococcus aureus isolated from bovine subclinical mastitis.

Chloramphenicol resistance (CmR) could be detected in 11 of 217 Staphylococcus aureus isolates from bovine subclinical mastitis. All isolates were assigned to biotypes A or C. The CmR-determinants were found to be located exclusively on small plasmids of approximately 4.6 kb as revealed by protoplast transformation. The 11 CmR-plasmids could be differentiated on the basis of restriction endonuclease analyses. The restriction maps of these CmR-plasmids identified two separate groups. One group demonstrated homology to the plasmid pC 221, the other to the plasmid pC 223. Both prototype plasmids, pC 221 and pC 223, had been isolated from S. aureus of human origin.     

Phage types, ribotypes and tetracycline resistance genes of Salmonella enterica subsp. enterica serovar Typhimurium strains isolated from different origins in Italy

The tetracycline resistance (tet) gene patterns of 52 tetracycline resistant Salmonella enterica subsp. enterica (S.) serovar Typhimurium isolates collected from animals, food of animal origin, and humans in Italy, were investigated to evaluate whether the tet gene patterns could be used for strain differentiation in addition to phage typing and ribotyping. The detection of tet genes was performed by specific PCR assays. Ribotyping was performed automatically using PvuII as restriction enzyme. Ten different ribotyping patterns were detected. All isolates were positive for at least one of the tet genes studied and six different tet gene patterns were observed. Ribotyping and tet gene patterns showed discriminatory indices of 0.741 and 0.812, respectively. Multiple tet genes were commonly found among tetracycline resistant S. typhimurium isolates from various sources. The resulting tet gene patterns allowed further discrimination of strains which were otherwise indistinguishable by their phage type, ribotype and origin. Thus, the analysis of tet gene patterns might represent an additional tool for the differentiation of S. typhimurium isolates.     

Effects of different media on proliferation and differentiation capacity of canine, equine and porcine adipose derived stem cells

Adult stem cells are of particular interest for therapeutic use in the field of regenerative medicine. Adipose-derived mesenchymal stem cells (ASCs) are an attractive stem cell source for all fields of regenerative medicine because adipose tissue - and therewith cells - can easily be harvested from each donor. However, common expansion using fetal bovine serum (FBS) can not be used for clinical applications as xenogenic proteins must be avoided. Adipose tissue from equine, canine and porcine donors was digested with collagenase to isolate ASCs. ASCs were either expanded in a cell culture medium supplemented with FBS or in a serum-free medium (UltraCulture; UC) supplemented with a serum substitute (UltroserG). From all three animal species, the adipogenic and osteogenic differentiation potential of ASCs cultured with different media was analyzed in vitro. Cell proliferation analysis showed a population doubling time of 48-68 h for canine cells, 54-65 h for porcine cells and 54-70 h for equine cells, expanded in different media. Except for porcine ASCs, cells cultured in media supplemented with FBS grew faster than cells expanded in UC medium with UltroserG. Yet, all cells maintained their potential to differentiate into adipocytes and osteoblasts. UltraCulture medium containing UltroserG can for all examined species be recommended if FBS needs to be avoided in the expansion of donor-derived (stem) cells.     

Nonsuppurative encephalitis in a dog

A 4-year-old male German Hunting Terrier presented with tremor, dyspnea, trismus, spasms of the musculature of the larynx and pharynx, and hypothermia and subsequently died despite intensive clinical care. Prior clinical signs included vomitus and diarrhea. Microscopic examination of the brain revealed a multifocal nonsuppurative brain stem encephalitis; a few intralesional neurons contained intranuclear inclusions. By immunohistochemistry, Aujeszky disease virus (Suid herpesvirus 1) antigen was detected in neurons in the brain and in ganglion cells of the trigeminal ganglia. Viral culture of brain tissue confirmed the presence of Aujeszky disease virus. Histopathologic findings in the brain with the identification of Aujeszky disease virus by immunohistochemistry and polymerase chain reaction are consistent with Aujeszky disease virus-induced encephalitis. Sequencing revealed a 100% homology of the isolated Aujeszky disease virus with Aujeszky disease virus isolates of wild boar from Eastern Germany.     

Effect of a rumen-protected conjugated linoleic acid mixture on hepatic lipid metabolism in heifers

This study was performed to assess the effects of rumen-protected conjugated linoleic acid (CLA) on hepatic lipid metabolism in heifers. In particular, it was of interest whether feeding CLA causes development of fatty liver as observed recently in mice. Thirty-six growing heifers with an initial body weight of 185 kg were allotted to three treatment groups and fed daily 250 g of different rumen-protected fats for 16 weeks: The control group received 250 g of a CLA-free control fat, the CLA100 group received 100 g of a CLA fat containing 2.4% of cis-9, trans-11 CLA and 2.1% of trans-10, cis-12 CLA and 150 g control fat and the CLA250 group received 250 g of the CLA fat. CLA supplementation had no effect on animal performance parameters, liver weight and hepatic triglyceride concentration. Moreover, mRNA expression of hepatic genes involved in lipogenesis, β-oxidation and fatty acid transport was not influenced by dietary CLA. The fatty acid composition of hepatic total lipids, with particular consideration of ratios of fatty acids indicative of Δ9-, Δ6- and Δ5-desaturation, was also less influenced by dietary CLA. In conclusion, the study shows that dietary rumen-protected CLA has less effect on hepatic lipid metabolism in young heifers and does not induce the development of a fatty liver such as in mice.     

Diagnostic value of the neutrophil myeloperoxidase index in horses with systemic inflammation

The myeloperoxidase index (MPXI) was investigated as a diagnostic indicator of systemic inflammation in a retrospective study using data from 859 hospitalised horses. A reference interval of 8.5-10.4 for the MPXI was established. In horses with systemic inflammatory response syndrome (SIRS), the MPXI was significantly lower than in healthy horses, those with localised inflammation and those with sepsis. The MPXI in horses with sepsis was also significantly lower than in healthy animals and those with localised inflammation. Horses in the SIRS group with leucopenia, white blood cell (WBC) count within the reference interval (WRI) or leucocytosis had significantly lower MPXIs than healthy horses, those with localised inflammation and those with sepsis in the same WBC count subgroups. In horses with sepsis and WBC count WRI, the MPXI was significantly lower than in healthy horses or those with localised inflammation. MPXI is a useful complementary tool to identify horses with systemic inflammation, especially if they have WBC counts WRI.     

COMPUTED TOMOGRAPHIC FEATURES OF BASIHYOID ECTOPIC THYROID CARCINOMA IN DOGS

Eight dogs with a firm, nonpainful swelling in the ventral laryngeal region and with a final diagnosis of ectopic thyroid carcinoma were investigated by Computed Tomography (CT) at six different institutions. Computed Tomography findings were reviewed, focusing on lesion volume, shape, margins, relationship with surrounding structures and adjacent vessels, attenuation characteristics, and presence of metastases. Ectopic thyroid carcinomas were seen as oval‐to‐bilobed masses centered on the basihyoid bone with associated bone lysis, highly vascularized capsules with central poorly contrast enhancing areas. In all cases there was laryngeal wall infiltration, in two dogs invasion of the laryngeal lumen and in one case invasion of the ventral muscular and subcutaneous plane. Metastases were found in retropharyngeal lymph nodes (three cases) and in the lung (two cases). Ectopic thyroid carcinoma should be considered in the differential diagnosis when a mass in the basihyoid region is present. Described CT features may be typical for ectopic thyroid neoplasia and could be used to help decide the therapeutic plan.     

Use of isotope-labeled aflatoxins for LC-MS/MS stable isotope dilution analysis of foods

Aflatoxins are a group of very carcinogenic mycotoxins that can be found on a wide range of food commodities including nuts, cereals, and spices. In this study, the first LC-MS/MS stable isotope dilution assay (SIDA) for the determination of aflatoxins in foods was developed. The development of this method was enabled by easily accessible isotope-labeled (deuterated) aflatoxins B2 and G2, which were synthesized by catalytic deuteration of aflatoxin B1 and G1, purified, and well-characterized by NMR and MS. All four aflatoxins of interest (B1, B2, G1, and G2) were quantified in food samples by using these two labeled internal standards. The response factors (RF) of the linear calibrations were revealed to be matrix independent for labeled aflatoxin B2/aflatoxin B2 and labeled aflatoxin G2/aflatoxin G2. For labeled aflatoxin B 2/aflatoxin B 1 and labeled aflatoxin B2/aflatoxin G1 matrix-matched calibration was performed for the model matrices almonds and wheat flour, showing significant differences of the RFs. Limits of detection (LOD) were determined by applying a statistical approach in the presence of the two model matrices, yielding 0.31 microg/kg (aflatoxin B1), 0.09 microg/kg (aflatoxin B2), 0.38 microg/kg (aflatoxin G1), and 0.32 microg/kg (aflatoxin G2) for almonds (similar LODs were obtained for wheat flour). Recovery rates were between 90 and 105% for all analytes. Coefficients of variation (CV) of 12% (aflatoxin B1), 3.6% (aflatoxin B2), 14% (aflatoxin G1), and 4.8% (aflatoxin G2) were obtained from interassay studies. For further validation, a NIST standard reference food sample was analyzed for aflatoxins B1 and B2. The method was successfully applied to determine trace levels of aflatoxins in diverse food matrices such as peanuts, nuts, grains, and spices. Aflatoxin contents in these samples ranged from about 0.5 to 6 microg/kg.