Serum resistance and the traT gene in bovine mastitis-causing Escherichia coli

Ninety-five Escherichia coli isolates from bovine mastitis, 47 isolates from milking machine filters, 36 enterotoxigenic (ETEC) and 43 verocytotoxigenic (VTEC) isolates from cows were examined for the ability to resist the bactericidal effects of 90% gnotobiotic calf serum. There was no significant difference in the percentage of isolates in each group which demonstrated resistance. Two potential virulence traits, the traT gene and the K1 capsular antigen, previously shown to be related to serum resistance, in human E. coli pathogens, were also examined. Using colony blot hybridization there was no significant difference in the percentage of isolates in each group carrying the traT gene. A significant relationship between the presence of the traT gene and serum resistance was not found in any of the four groups of E. coli isolates tested. Only 3.2% of the bovine mastitis, 2.1% of the milk filter and 4.6% of the VTEC isolates were positive for the K1 capsular antigen. Again, no correlation between either the K1 antigen and serum resistance or between the K1 antigen and the presence of the traT gene was found in any of the four groups. None of the antimicrobial resistance patterns of the isolates were the same as those demonstrated by R plasmids known to carry the traT gene. Thus, it appears that the traT gene may not be related to serum resistance in bovine E. coli isolates.     

Performance characteristics of methods of analysis used for regulatory purposes. I. Drug dosage forms. E. miscellaneous methods

Precision parameters of miscellaneous methods for the analysis of drug dosage forms approved by AOAC since 1972, and not previously reviewed in this series, were recalculated on a consistent statistical basis by using the computer program FDACHEMIST. Seventeen published collaborative studies were reviewed; the studies encompassed 19 analytes in 80 different materials (dosage forms), 102 collaborative assays, approximately 10 laboratories per study, and principally direct spectrophotometric, polarographic, and spectroscopic methods, for a total of 1451 determinations. The average repeatability relative standard deviation (within-laboratories, RSDo) for the instrumental methods was 1.5%; the reproducibility relative standard deviation (among-laboratories, including within-, RSDx) was 2.6%; the ratio RSDo/RSDx of the averages was 0.57, with an average outlier rate of 2.7% of the reported determinations. The line of best fit of RSDx for the instrumental methods plotted against the negative logarithm of the concentration increases slightly with decreasing concentration, extending from an RSDx of approximately 2.0% at 100% concentration to an RSDx of 3.4% at 0.001% (10 ppm) concentration; this represents an RSDx change of approximately 0.3% (absolute) for each 10-fold decrease in concentration, independent of analyte, matrix, and method. A method for determining precipitated allergenic protein by the micro-Kjeldahl technique appeared to be outside this general relation, showing an RSDx of about 13% at a concentration of 0.015% (150 ppm) nitrogen.     

Feasibility of pooled-sample testing for the detection of porcine reproductive and respiratory syndrome virus antibodies on serum samples by ELISA

Surveillance of porcine reproductive and respiratory syndrome (PRRS) in negative sow farms is usually performed by testing for the presence of antibodies against PRRS virus in serum with a commercial ELISA test. The objective of this study was to evaluate the feasibility of pooling serum samples for detection of PRRS virus antibodies by ELISA. The effect of pool size on the sensitivity and specificity of the ELISA test was evaluated by testing true positive samples and false positive samples, respectively, diluted in negative sera. The effect of three different cut-off values for the interpretation of the diagnostic test (0.4, 0.3 and 0.2) was evaluated as well. Furthermore, the obtained sensitivity and specificity estimates were used to calculate the herd sensitivity and herd specificity of surveillance protocols in different scenarios. The results showed that pooling serum samples to detect PRRSV antibodies resulted in a decrease in sensitivity and an increase in specificity, compared to testing individual samples, while the reduction of the s/p cut-off value recommended by the manufacturer (0.4) had the opposite effect. We describe an approach that can increase the herd sensitivity of a surveillance protocol for breeding herds, while maintaining high herd specificity and low testing costs. This can be achieved by sampling a larger number of animals and running the samples in pools. Therefore, the conventional monitoring protocols based on ELISA on individual samples can be improved by using pooled-sample testing.     

Detection of foot-and-mouth disease virus by nucleic acid sequence-based amplification (NASBA)

A study was conducted to evaluate the performance of a nucleic acid sequence-based amplification (NASBA) assay for the detection of foot-and-mouth disease virus (FMDV). Two detection methods: NASBA-electrochemiluminescence (NASBA-ECL) and a newly developed NASBA-enzyme-linked oligonucleotide capture (NASBA-EOC) were evaluated. The diagnostic sensitivity of these assays was compared with other laboratory-based methods using 200 clinical samples collected from different regions of the world. Assay specificity was also assessed using samples (n=43) of other viruses that cause vesicular disease in livestock and genetic relatives of FMDV. Concordant results were generated for 174/200 (87.0%) of suspect FMD samples between NASBA-ECL and real-time RT-PCR. In comparison with the virus isolation (VI) data, the sensitivity of the NASBA-ECL assay was 92.9%, which was almost identical to that of the real-time RT-PCR (92.4%) for the same set of samples. There was broad agreement between the results of the NASBA-ECL and the simpler NASBA-EOC detection method for 97.1% of samples. In conclusion, this study provides further data to support the use of NASBA as a rapid and sensitive diagnostic method for the detection and surveillance of FMDV.     

Differentiation and characterization of rat adipose tissue mesenchymal stem cells into endothelial‐like cells

In this study, mesenchymal stem cells were isolated from rat adipose tissue (AD‐MSCs) to characterize and differentiate them into endothelial‐like cells. AD‐MSCs were isolated by mechanical and enzymatic treatments, and their identity was verified by colony‐forming units (CFU) test and by differentiation into cells of mesodermal lineages. The endothelial differentiation was induced by plating another aliquot of cells in EGM‐2 medium, enriched with specific endothelial growth factors. Five subcultures were performed. The expression of stemness genes (OCT4, SOX2 and NANOG) was investigated. The presence of CD90 and the absence of the CD45 were evaluated by flow cytometry. The endothelial‐like cells were characterized by the evaluation of morphological changes and gene expression analysis for endothelial markers (CD31, CD144, CD146). Characterization of AD‐MSCs showed their ability to form clones, to differentiate in vitro and the OCT‐4, SOX‐2, NANOG genes expression. Immunophenotypic characterization showed the CD90 presence and the CD45 absence. The endothelial‐like cells showed morphological changes, the expression of CD31, CD144, CD146 genes and the presence of CD31 membrane receptor. Matrigel assay showed their ability to form network and vessels‐like structures. This study lays the foundations for future evaluation of the potential AD‐MSCs pro‐angiogenic and therapeutic role.