CLS advanced degrees and career enhancement. Part 1--Comparison of career data.

OBJECTIVE: Determine whether recipients of clinical laboratory science (CLS) advanced degrees (MS) experience greater career achievements than their baccalaureate level (BS) colleagues. DESIGN: Two similar questionnaires were used-one for certified or licensed CLS professionals who had earned advanced CLS degrees (MS); the other for matched BS CLS colleagues. SETTING: Five academic programs that conduct both National Accrediting Agency for Clinical Laboratory Sciences accredited CLS education and CLS MS degree programs participated. PARTICIPANTS: The number of survey respondents was 220, 117 with advanced CLS degrees and 103 BS level controls. There were 99 matched pairs, i.e., 198 individuals were matched for gender, residence region, and years of experience. MAIN OUTCOME MEASURES: Careers of BS vs. MS respondents were statistically compared, e.g., fractions with managerial level jobs, relative earnings increases per year, numbers of publications and reports, and other professional contributions. RESULTS: Compared to their BS degree controls, MS degree respondents had more managerial level jobs (62% MS; 36% BS), a higher frequency of job change (once per 4.3 years MS; once per 5.9 years BS), and a higher increase per year of earnings (9.1% MS; 8.1% BS). A greater percentage of the MS degree graduates (77%) than the BS level controls (33%) had authored external publications; the responses related to authorship of institutional reports and procedures were less different-84% MS and 64% BS. Professional contributions to their institutions or profession were cited slightly more frequently by the MS graduates (65%) than by the BS level controls (55%). CONCLUSION: Compared to their matched BS level CLS colleagues, CLS MS degree recipients had greater job mobility, greater management authority, higher salary, and more numerous professional contributions.

CLS advanced degrees and career enhancement. Part 2--A comparison of perceptions.

OBJECTIVE: To determine whether recipients of clinical laboratory science (CLS) advanced degrees (MS) perceive greater career enhancement value related to earning an advanced degree than is perceived by their baccalaureate level (BS) colleagues. DESIGN: Two questionnaires were used-one for certified or licensed CLS professionals who had earned MS CLS degrees; the other for matched BS CLS colleagues. SETTING: Five academic programs that conduct both National Accrediting Agency for Clinical Laboratory Sciences accredited CLS education and CLS MS degree programs participated. PARTICIPANTS: The number of survey respondents was 220 (117-MS; 103-BS level controls). The groups were matched for gender, residence region, and years of experience. MAIN OUTCOME MEASURES: The primary outcome measurements were the perceived benefits of having a CLS MS degree, the reasons for and against obtaining a CLS MS degree, and the overall evaluation of CLS degree programs at both levels. RESULTS: The highest perceived benefit of having a CLS MS degree was the same in both groups, "enhanced self esteem and confidence". The highest priority motivation of MS degree recipients for obtaining a CLS advanced degree was "personal satisfaction". The highest priority reason of the BS group for not obtaining a CLS advanced degree was "family obligation". In both levels of degree programs the subject most commonly cited as needing modification was laboratory management. CONCLUSION: The results indicate that CLS professionals who have CLS MS degrees perceive a greater career enhancement value of advanced CLS degrees than their BS level colleagues.

Prevalence of human herpesvirus 6 in lung tissue from children with pneumonitis.

AIMS/BACKGROUND: Human herpesvirus type 6 (HHV-6) is the aetiological agent of exanthem subitum, and has also been linked with a variety of other diseases. The aim of this study was to investigate the role of HHV-6 in pneumonitis in children. METHODS: Formalin fixed, paraffin wax embedded lung tissue from 33 children (age range two months to 16 years) who died with pneumonitis was subjected to immunohistochemical staining for HHV-6 using an avidin-biotin method. RESULTS: Active HHV-6 infection was demonstrated in four children: a bone marrow transplant recipient with concomitant adenovirus infection, a patient with hepatitis of unknown aetiology, a patient with congenital anomalies, and a patient with congenital immunodeficiency. CONCLUSION: Accurate localisation of HHV-6 is possible in postmortem lung tissue. HHV-6 either alone or in combination with other pathogens may play a role in the development of pneumonitis.

Augmentation of macrophage phagocytic activity by cell-free extracts of selected lactic acid-producing bacteria.

Oral and intraperitoneal administration of lactic acid-producing bacteria can significantly augment the immune response in murine models; however, the immunopotentiating effects in these studies differ significantly. Murine macrophagelike cell line J774 was cultured in the presence of cell-free extracts of Lactobacillus acidophilus and Bifidobacterium longum, and the effect on macrophage function was evaluated by measurement of synthesis of selected enzymes and their ability to take up either acrylamide particles or live Salmonella typhimurium. Lysozyme activity of J774 cells was significantly decreased by cell-free extracts of B. longum, but not of L. acidophilus, whereas extracts of both strains induced morphological changes and significantly enhanced phagocytosis of inert particles or viable Salmonella. Whole cell extracts of lactic acid-producing bacteria are therefore capable of altering macrophage function in a strain-dependent manner.

Inability to detect mycobactin in mycobacteria-infected tissues suggests an alternative iron acquisition mechanism by mycobacteria in vivo.

Although most species of mycobacterium are capable of producing mycobactin, it is not known if conditions within the host allow for mycobactin synthesis or whether it even plays a role in iron acquisition in vivo. We employed the mycobactin-auxotroph, Mycobacterium paratuberculosis, in a bioassay to examine tissues from animals infected with either Mycobacterium tuberculosis, Mycobacterium avium or M. paratuberculosis for the presence of mycobactin or compounds which demonstrate mycobactin-like activity. Other iron-binding compounds, including purified siderophores from unrelated organisms and host iron-binding proteins were also evaluated in the bioassay for growth induction of M. paratuberculosis in the absence of mycobactin. Although mycobactin could be easily demonstrated in tissues artificially seeded with mycobacteria, no mycobactin could be detected in heavily infected tissues. None of the purified siderophores from unrelated microorganisms were found to support growth of M. paratuberculosis in the absence of mycobactin. Host iron-binding proteins (transferrin, lactoferrin, ferritin, hemin) also failed to induce growth in the bioassay at pH 6.8, however, when the pH was adjusted between 5-6.2, transferrin and lactoferrin promoted growth of M. paratuberculosis without mycobactin, probably as a result of the dissociation of iron rather than a specific interaction. We confirm that mycobacteria are incapable of iron uptake when iron is chelated to siderophores from unrelated organisms and conclude that mycobactin-mediated mechanisms of iron-acquisition by mycobacteria do not appear to have as significant a role in vivo as in vitro. In addition, evidence is presented that suggests iron-containing transferrin and lactoferrin at low pH may circumvent the need for mycobactin by M. paratuberculosis.

Mycobacterium paratuberculosis. Factors that influence mycobactin dependence.

Mycobacterium paratuberculosis does not produce any detectable mycobactin, an iron-binding compound that is synthesized by most Mycobacterium spp. and necessary for the growth of all mycobacteria. This study examined the influence of various culture conditions on mycobactin dependence in M. paratuberculosis. Using a radiometric growth assay, we found the minimal concentration of mycobactin J necessary for growth of M. paratuberculosis to be 0.006 microM, whereas 1.2 microM (1 microgram/ml) was required for optimal growth. In media without mycobactin at iron concentrations less than or equal to 100 microM, growth of M. paratuberculosis occurred at pH 5.0, but not pH 6.8. Iron concentrations greater than 100 microM did not significantly increase growth at pH 5.0, but at pH 6.8 the growth rate increased with increasing amounts of iron reaching a rate equal to control cultures containing mycobactin. Mycobacterium paratuberculosis appeared to lose mycobactin dependence when subcultured; however, this was subsequently shown to be a result of mycobactin carried over from primary medium. Removal of this contaminating cell-wall-associated mycobactin reestablished mycobactin dependence. We conclude that mycobactin dependence must be carefully determined because it is a key test used in identification of M. paratuberculosis and may be easily influenced by media pH, iron concentration, and mycobactin carryover from primary media.

Crossed immunoelectrophoretic analysis of Mycobacterium paratuberculosis.

Antigenic analysis of M. paratuberculosis revealed extensive cross-reactivity with M. avium; however, the number of cross-reactive antigens found was dependent on the strain of M. avium tested. One antigen was shown to be the common antigen while another appeared to be iron-regulated in its production. A commercial polyclonal antibody to M. paratuberculosis produced a CIE precipitin pattern comparable to that of the antibody produced for the present study. An antigen designated no. 6 was consistently precipitated by sera from cattle infected with M. paratuberculosis. This antigen exhibited complete cross-reaction with M. avium and partial cross-reaction with M. phlei. Among three commercially available complement fixation (CF) antigen that could be precipitated by M. paratuberculosis antibodies. A commercial antigen for use in an agar gel immunodiffusion test for Johne's disease diagnosis produced 12 precipitins with the M. paratuberculosis antibody, one of which was identical with antigen 6.

Effects of colonial morphology and tween 80 on antimicrobial susceptibility of Mycobacterium paratuberculosis.

Smooth (Sm) and rough (Rg) colonial types of Mycobacterium paratuberculosis ATCC 19698 and two clinical isolates were tested to examine their growth responses in medium containing antimicrobial agents. Susceptibility tests were done in Middlebrook 7H12B medium with and without Tween 80 and one of the following antimicrobial agents: streptomycin, isoniazid, rifampin, ethambutol, ciprofloxacin, and penicillin G. Growth responses in the presence of antimicrobial agents led to the following observations. (i) In the absence of Tween, Rg colony types were more resistant than Sm colony types; (ii) the addition of Tween 80 significantly increased the susceptibility of both Sm and Rg colony types; however, the increase was greater with the Sm colony types. These studies showed that the antimicrobial susceptibility of M. paratuberculosis was significantly affected when Tween 80 was present in either the primary culture medium or the drug susceptibility test medium. In the absence of the perturbing influence of Tween 80, M. paratuberculosis was resistant to the antimicrobial agents tested.

Enhanced radiometric detection of Mycobacterium paratuberculosis by using filter-concentrated bovine fecal specimens.

A commercial radiometric medium, BACTEC 12B, was modified by addition of mycobactin, egg yolk suspension, and antibiotics (vancomycin, amphotericin B, and nalidixic acid). Decontaminated bovine fecal specimens were filter concentrated by using 3-microns-pore-size, 13-mm-diameter polycarbonate filters, and the entire filter was placed into the radiometric broth. Comparison of the radiometric technique with conventional methods on 603 cattle from 9 Mycobacterium paratuberculosis-infected herds found that of 75 positive specimens, the radiometric technique detected 92% while conventional methods detected 60% (P less than 0.0005). Only 3.9% of radiometric cultures were contaminated. To measure the effect of filter concentration of specimens on the detection rate, 5 cattle with minimal and 5 with moderate ileum histopathology were sampled weekly for 3 weeks. M. paratuberculosis was detected in 33.3% of nonfiltered specimens and 76.7% of filtered specimens (P less than 0.005). Detection rates were directly correlated with the severity of disease, and the advantage of specimen concentration was greatest on fecal specimens from cattle with low-grade infections. Detection times were also correlated with infection severity: 13.4 +/- 5.9 days with smear-positive specimens, 27.9 +/- 8.7 days with feces from cows with typical subclinical infections, and 38.7 +/- 3.8 days with fecal specimens from cows with low-grade infections. Use of a cocktail of vancomycin, amphotericin B, and nalidixic acid for selective suppression of nonmycobacterial contaminants was better than the commercial product PANTA (Becton Dickinson Microbiologic Systems, Towson, Md.) only when specimens contained very low numbers of M. paratuberculosis. Radiometric culture of filter-concentrated specimens generally doubled the number of positive fecal specimens detected over conventional methods, making it a useful tool for diagnosis and control of bovine paratuberculosis.

Effect of polyoxyethylene sorbate compounds (Tweens) on colonial morphology, growth, and ultrastructure of Mycobacterium paratuberculosis.

Polyoxyethylene sorbate (Tween) compounds were tested to compare their growth stimulation effects on Mycobacterium paratuberculosis. Three low passage and three high passage clinical isolates and ATCC strain 19698 were used. Tween 20, 40, 60, and 80 were tested at concentrations of 0, 0.001, 0.01, 1.0, and 3.0% (w/v) in radiometric broth culture media and in Middlebrook 7H9 agar plates. Scanning and transmission electron microscopy were used to examine cell wall appearance and ultrastructure, respectively. In broth culture, 0.1% (w/v) Tween 60 most dramatically enhanced growth of M. paratuberculosis ATCC strain 19698. The effects of Tween 40 and 80 on growth took a bimodal form, enhancing growth at concentration ranges of 0-0.01% and 0.1-1.0% (w/v) but suppressing growth at concentrations of 0.01-0.1% (w/v). Two of three high passage clinical isolates grew optimally in the presence of 1.0% (w/v) Tween 80, while the remaining high passage isolate and all three low passage isolates grew best in media containing 0.1% (w/v) Tween 80. Colonial morphology of all strains grown on Middlebrook 7H9 agar without Tween 80 was irregular and granular whereas colonies on plate media containing greater than 0.01% (w/v) Tween 80 were entire, smooth, and domed. Scanning electron microscopy also revealed a transition from rough to smooth cell walls with increasing Tween 80 concentration. Transmission electron microscopy showed the presence of low electron dense intracellular vacuoles in Tween 80 grown M. paratuberculosis cells. Thus, Tweens altered colonial morphology, the cell wall surface, and ultrastructure of M. paratuberculosis and stimulated its growth in vitro in a concentration-dependent, and often bimodal, fashion.(ABSTRACT TRUNCATED AT 250 WORDS)

A model for analyzing growth kinetics of a slowly growing Mycobacterium sp.

This report describes a simple method for quantifying viable mycobacteria and for determining generation time. We used statistical models and computer analysis of growth curves generated for the slowly growing mycobacterium Mycobacterium paratuberculosis under controlled conditions to derive a mathematical formula relating the dependent variable, growth, to the independent variables, log10 number of organisms in the inoculum (inoculum size) and incubation time. Growth was measured by a radiometric method which detects 14CO2 release during metabolism of a 14C-labeled substrate. The radiometric method allowed for early detection of growth and detected as few as three viable bacteria. The coefficient of variation between culture vials inoculated with the same number of M. paratuberculosis was 0.083. Radiometric measurements were highly correlated to spectrophotometric and plate count methods for measuring growth (r = 0.962 and 0.992, respectively). The proportion of the total variability explained by the model in a goodness of fit test was 0.9994. Application of the model to broth cultures provided accurate estimates of the number of M. paratuberculosis (standard error = 0.21, log10 scale) and the growth rate (coefficient of variation, 0.03). Generation time was observed to be dependent upon the number of organisms in the inoculum. The model accurately described all phases of growth of M. paratuberculosis and can likely be applied to other slowly growing microorganisms.