Bovine mastitis is the most important source of loss for the dairy industry. A rapid and specific test for the detection of the main pathogens of bovine mastitis is not actually available. Molecular probes reacting in PCR with bacterial DNA from bovine milk, providing direct and rapid detection of Escherichia coli, Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus parauberis, and Streptococcus uberis, have been developed. Two sets of specific primers were designed for each of these microorganisms and appeared to discriminate close phylogenic bacterial species (e.g., S. agalactiae and S. dysgalactiae). In addition, two sets of universal primers were designed to react as positive controls with all major pathogens of bovine mastitis. The sensitivities of the test using S. aureus DNA extracted from milk with and without a pre-PCR enzymatic lysis step of bacterial cells were compared. The detection limit of the assay was 3.125 ؋ 10 2 CFU/ml of milk when S. aureus DNA was extracted with the pre-PCR enzymatic step compared to 5 ؋ 10 3 CFU/ml of milk in the absence of the pre-PCR enzymatic step. This latter threshold of sensitivity is still compatible with its use as an efficient tool of diagnosis in bovine mastitis, allowing the elimination of expensive reagents. The two PCR tests avoid cumbersome and lengthy cultivation steps, can be performed within hours, and are sensitive, specific, and reliable for the direct detection in milk of the six most prevalent bacteria causing bovine mastitis.Bovine mastitis (BM) is an inflammation of the mammary gland, usually due to a microbial infection (28), which causes North American dairy producers to lose billions of dollars every year. These losses are primarily due to lower milk yields, reduced milk quality, and higher production costs. BM often becomes chronic, and it is important to identify quickly the new clinical cases in order to control infection in the herd. The bacteria responsible for BM can be classified as environmental (Escherichia coli, Streptococcus dysgalactiae, Streptococcus parauberis, and Streptococcus uberis) or contagious (Staphylococcus aureus and Streptococcus agalactiae) depending of their primary reservoir (environment versus infected mammary gland quarter) (11,25).The suitability of a detection method for routine diagnosis depends on several factors, such as specificity, sensitivity, expense, amount of time, and applicability to large numbers of milk samples. The most common but unspecific method (2) to identify potential chronic infections is a somatic cell count: the California Mastitis Test in field conditions and the automated method in the diagnosis laboratory. Currently, the method of identification of the mammary gland pathogens is by in vitro culture, which provides the "gold standard"; however, this technique is labor-intensive and time-consuming. Two other problems can be encountered when these methods of identification are used: first, 2 to 3 days are required to grow, isolate, and identify the pathoge...

The in vitro activities of 10 families of antimicrobial agents alone and in combination with a synthetic polycationic polymer, polyethylenimine (PEI), against a resistant clinical isolate of Pseudomonas aeruginosa were investigated by MIC assays, checkerboard testing, and killing curve studies. At a concentration of 250 nM, PEI (10 kDa) was not directly bactericidal or bacteriostatic; but when it was used in combination with novobiocin, ceftazidime, ampicillin, ticarcillin, carbenicillin, piperacillin, cefotaxime, chloramphenicol, rifampin, or norfloxacin, it significantly reduced the MICs of these antibiotics by 1.5-to 56-fold. However, the MICs of aminoglycosides, polymyxins, and vancomycins were increased by 1.2-to 5-fold when these drugs were combined with PEI; and the MICs of tetracycline, erythromycin, ciprofloxacin, and ofloxacin were not affected when these drugs were combined with PEI. In the killing curve studies, combinations of PEI with novobiocin, ceftazidime, chloramphenicol, or rifampin resulted in 5-to 8-log 10 CFU/ml reductions in bacterial counts when 25% of the MIC of each antibiotic was used. These results indicate that infections due to resistant Pseudomonas strains could be treated by the use of a synergistic combination of PEI and antimicrobial drugs.Over the past two decades, Pseudomonas aeruginosa has attracted attention as an opportunistic pathogen in hospitalized, immunocompromised, and cystic fibrosis patients (6,15,20). Despite aggressive antibiotic therapy, P. aeruginosa is rarely eradicated owing to its high level of intrinsic resistance to many drugs (14,19,27). This resistance is due to the effective permeability of the outer membrane (OM) of gram-negative bacteria to both hydrophobic antibiotics and high-molecular-weight hydrophilic drugs (for reviews, see references 4, 25, 28, and 30). Unlike most cell types, gram-negative bacteria surround themselves with a double membrane. The outermost of these two membranes is asymmetric, with the inner leaflet composed of glycerophospholipids and the outer leaflet predominantly composed of lipopolysaccharide (LPS). Tight interactions between the highly negatively charged LPS molecules are believed to form an effective barrier against hydrophobic compounds. For these polyanionic molecules to form a stable "tiled roof" on the surface of the OM, adjacent LPS molecules are linked electrostatically by divalent cations (Ca 2ϩ , Mg 2ϩ ) present in the OM. Polyethylenimine (PEI) is a weakly basic, aliphatic, nontoxic synthetic polymer which is polycationic owing to the presence of primary, secondary, and tertiary amino groups. It is well known that certain polycationic agents such as polymyxin and its derivatives polylysines and protamine can increase the permeability of the gram-negative bacterial OM to solutes that are normally unable to penetrate (25). Helander et al. found that, when it was applied alone, PEI has a strong permeabilizing effect but no bactericidal effect on gram-negative bacteria (8). Escherichia coli, P. aeruginosa, and Salmon...

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Abstract.As biomarkers continue to become an integral part of drug development and decision-making, there are increased expectations for reliable and quantitative assays. Protein biomarker assay results are directly influenced by the calibrator material. The selection of calibrator material presents many challenges that impact the relative accuracy and performance of the assay. There is an industry-wide challenge finding reliable and wellcharacterized calibrator material with good documentation. Several case studies are presented that demonstrate some of the challenges involved in selecting appropriate calibrators along with the resolutions that were ultimately applied. From these experiences, we present here a set of recommendations for selecting and characterizing calibrator material based on the intended purpose of the assay. Finally, we introduce a commutability approach, based on common clinical chemistry practices, which can be used to demonstrate interchangeability with calibrator materials across multiple lots and technology platforms for all types of protein biomarker assays.

The Canadian federal government instituted an environment effects monitoring (EEM) program for the pulp and paper industry to ensure the long-term integrity of fish populations below the discharge of mill effluents. The program is cyclical in nature (every three to four years) and one of the components of the program is an adult fish survey which monitors wild fish health in the receiving environments. Cycle 1 reports were submitted in April of 1996, and the report for the James Maclaren Inc. mill at Thurso, Quebec, suggested some responses were evident in fish downstream of the mill discharge. However, the receiving environment was subject to inputs from other potential sources or confounding factors such as farm runoff, storm sewers, municipal discharges and and upstream paper mills. A review of other Cycle 1 studies also identified a number of problems encountered during the adult fish surveys. At this time, Maclaren researchers and Environment Canada set up a collaborative research project as part of their Cycle 2 studies at the Thurso site. Objectives of the study included a comparison of Cycle 2 results to their Cycle 1 studies, an assessment of the potential for the use of forage fish in the EEM program as well as an evaluation of reference site variability in the required EEM fish parameters. Collections in 1997 confirmed an increased male yellow perch liver size downstream of the discharge, but could not confirm reproductive alterations in exposed fish. It was possible to obtain all of the required EEM fish parameters from a small forage fish species such as the johnny darter, which may reduce the influence of fish mobility and other confounding factors on fish responses at some sites. Reference site variability was not a contributing factor in evaluating fish responses to effluent sources at this site.