Effects of season, age, sex, and housing on salivary cortisol concentrations in horses.

Analysis of salivary cortisol is increasingly used to assess stress responses in horses. Because spontaneous or experimentally induced increases in cortisol concentrations are often relatively small for stress studies, proper controls are needed. This requires an understanding of the factors affecting salivary cortisol over longer times. In this study, we have analyzed salivary cortisol concentration for 6 mo in horses (n = 94) differing in age, sex, reproductive state, and housing. Salivary cortisol followed a diurnal rhythm with the highest concentrations in the morning and a decrease throughout the day (P < 0.001). This rhythm was disrupted in individual groups on individual days; however, alterations remained within the range of diurnal changes. Comparison between months showed highest cortisol concentrations in December (P < 0.001). Cortisol concentrations increased in breeding stallions during the breeding season (P < 0.001). No differences in salivary cortisol concentrations between nonpregnant mares with and without a corpus luteum existed. In stallions, mean daily salivary cortisol and plasma testosterone concentrations were weakly correlated (r = 0.251, P < 0.01). No differences in salivary cortisol between female and male young horses and no consistent differences between horses of different age existed. Group housing and individual stabling did not affect salivary cortisol. In conclusion, salivary cortisol concentrations in horses follow a diurnal rhythm and are increased in active breeding sires. Time of the day and reproductive state of the horses are thus important for experiments that include analysis of cortisol in saliva.

Multi-center evaluation of the VITEK® MS system for mass spectrometric identification of non-Enterobacteriaceae Gram-negative bacilli.

Studies have demonstrated that matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a rapid, accurate method for the identification of clinically relevant bacteria. The purpose of this study was to evaluate the performance of the VITEK MS v2.0 system (bioMérieux) for the identification of the non-Enterobacteriaceae Gram-negative bacilli (NEGNB). This multi-center study tested 558 unique NEGNB clinical isolates, representing 18 genera and 33 species. Results obtained with the VITEK MS v2.0 were compared with reference 16S rRNA gene sequencing and when indicated recA sequencing and phenotypic analysis. VITEK MS v2.0 provided an identification for 92.5 % of the NEGNB isolates (516 out of 558). VITEK MS v2.0 correctly identified 90.9 % of NEGNB (507 out of 558), 77.8 % to species level and 13.1 % to genus level with multiple species. There were four isolates (0.7 %) incorrectly identified to genus level and five isolates (0.9 %), with one incorrect identification to species level. The remaining 42 isolates (7.5 %) were either reported as no identification (5.0 %) or called "mixed genera" (2.5 %) since two or more different genera were identified as possible identifications for the test organism. These findings demonstrate that the VITEK MS v2.0 system provides accurate results for the identification of a challenging and diverse group of Gram-negative bacteria.

Multi-centre evaluation of mass spectrometric identification of anaerobic bacteria using the VITEK® MS system.

Accurate and timely identification of anaerobic bacteria is critical to successful treatment. Classic phenotypic methods for identification require long turnaround times and can exhibit poor species level identification. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is an identification method that can provide rapid identification of anaerobes. We present a multi-centre study assessing the clinical performance of the VITEK(®) MS in the identification of anaerobic bacteria. Five different test sites analysed a collection of 651 unique anaerobic isolates comprising 11 different genera. Multiple species were included for several of the genera. Briefly, anaerobic isolates were applied directly to a well of a target plate. Matrix solution (α-cyano-4-hydroxycinnamic acid) was added and allowed to dry. Mass spectra results were generated with the VITEK(®) MS, and the comparative spectral analysis and organism identification were determined using the VITEK(®) MS database 2.0. Results were confirmed by 16S rRNA gene sequencing. Of the 651 isolates analysed, 91.2% (594/651) exhibited the correct species identification. An additional eight isolates were correctly identified to genus level, raising the rate of identification to 92.5%. Genus-level identification consisted of Actinomyces, Bacteroides and Prevotella species. Fusobacterium nucleatum, Actinomyces neuii and Bacteroides uniformis were notable for an increased percentage of no-identification results compared with the other anaerobes tested. VITEK(®) MS identification of clinically relevant anaerobes is highly accurate and represents a dramatic improvement over other phenotypic methods in accuracy and turnaround time.

Reduced-size microchips for identification of horses: response to implantation and readability during a six-month period.

In this study, readability of reduced-size microchips in horses and the response to implantation were analysed. It was hypothesised that small microchips can be implanted stress-free but are less readable than larger microchips. Adult mares (n=40) were implanted with a reduced-size microchip (10.9×1.6 mm) at the left side of the neck (size of conventional microchips 11.4×2.2 mm). Microchips were identified with three different scanners (A, B, C) immediately, and at 6, 12 and 28 weeks after implantation. Twelve out of the 40 mares were submitted to microchip implantation and control treatments and cortisol, heart rate and heart rate variability (HRV) were determined. From the chip-bearing side of the neck, microchips were identified with all scanners in all horses at all times. From the contralateral side, correct readings were always 100 per cent with scanner C and with scanners A and B ranged between 60 and 100 per cent. Heart rate and HRV variable sd of beat-to-beat interval increased slightly (P<0.01) at microchip implantation and control treatment, but cortisol concentration did not increase. In conclusion, reduced-size microchips are highly reliable for identification of horses. Compared with conventional microchips, the reduction in size did not impair readability. Microchip implantation is no pronounced stressor for horses.

Identification of Enterobacteriaceae by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry using the VITEK MS system.

This multicenter study evaluated the accuracy of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry identifications from the VITEK MS system (bioMérieux, Marcy l'Etoile, France) for Enterobacteriaceae typically encountered in the clinical laboratory. Enterobacteriaceae isolates (n = 965) representing 17 genera and 40 species were analyzed on the VITEK MS system (database v2.0), in accordance with the manufacturer's instructions. Colony growth (≤72 h) was applied directly to the target slide. Matrix solution (α-cyano-4-hydroxycinnamic acid) was added and allowed to dry before mass spectrometry analysis. On the basis of the confidence level, the VITEK MS system provided a species, genus only, or no identification for each isolate. The accuracy of the mass spectrometric identification was compared to 16S rRNA gene sequencing performed at MIDI Labs (Newark, DE). Supplemental phenotypic testing was performed at bioMérieux when necessary. The VITEK MS result agreed with the reference method identification for 96.7% of the 965 isolates tested, with 83.8% correct to the species level and 12.8% limited to a genus-level identification. There was no identification for 1.7% of the isolates. The VITEK MS system misidentified 7 isolates (0.7 %) as different genera. Three Pantoea agglomerans isolates were misidentified as Enterobacter spp. and single isolates of Enterobacter cancerogenus, Escherichia hermannii, Hafnia alvei, and Raoultella ornithinolytica were misidentified as Klebsiella oxytoca, Citrobacter koseri, Obesumbacterium proteus, and Enterobacter aerogenes, respectively. Eight isolates (0.8 %) were misidentified as a different species in the correct genus. The VITEK MS system provides reliable mass spectrometric identifications for Enterobacteriaceae.

Enteric fever in a 6-year-old traveler caused by Salmonella enterica serotypes Typhi and Paratyphi A: laboratory detection strategies and treatment options.

We report the first pediatric case of enteric fever caused by Salmonella enterica serotypes Typhi and Paratyphi A. Mixed infections are infrequently reported, potentially because detection of two different Salmonella serotypes in blood cultures is technically challenging. We suggest laboratory strategies to aid in the recognition of mixed infections.

Roseomonas mucosa isolated from bloodstream of pediatric patient.

We report a case of catheter-related bacteremia associated with Roseomonas mucosa isolated from an immunocompromised pediatric patient with a history of multiple episodes of urinary tract infection and bacteremia.

Molecular basis of antagonism between K70E and K65R tenofovir-associated mutations in HIV-1 reverse transcriptase.

The K70E mutation in HIV-1 reverse transcriptase was observed in 10% of virologic non-responders of the abacavir/lamivudine/tenofovir arm of ESS30009, alone, or in mixtures with K65R by population sequencing. Clonal analysis of six ESS30009 K70E isolates failed to identify double mutants carrying K65R+K70E. Site-directed K70E mutants had a replication capacity of 97+/-29%, but only 2.4+/-0.9% for K65R+K70E and 0.01% for K65R+K70E+M184V mutants. K65R+K70E phenotypic fold changes for abacavir, lamivudine and tenofovir were comparable to reported values for K65R alone. In molecular dynamic simulations, the epsilon-amino group of K65 was positioned 2.7+/-0.1A from the gamma-phosphate of the dTTP ligand and stabilized the triphosphate. In the R65 mutant, this distance increased to 4.2+/-0.4A and the interaction energy with the ligand was less favorable, but the K70 epsilon-amino group was repositioned closer to the gamma-phosphate and had a more favorable interaction energy. In the double mutant, E70 could not stabilize the gamma-phosphate, resulting in a more severe defect. The net effect of the atomic-level changes in the double mutant may be to destabilize the pyrophosphate leaving group of the ligand, more severely affecting the catalytic rate of the polymerization reaction than the R65 single mutation.

Increasing prevalence of HIV-1 protease inhibitor-associated mutations correlates with long-term non-suppressive protease inhibitor treatment.

Treatment of human immunodeficiency virus type 1 with protease inhibitors (PIs) is associated with the emergence of resistance-associated mutations. Treatment-characterized datasets have been used to identify novel treatment-associated protease mutations. In this study, we utilized two large reference laboratory databases (>115,000 viral sequences) to identify non-established resistance-associated protease mutations. We found 20 non-established protease mutations occurring in 82% of viruses with a PI resistance score of 4-7, 62% of viruses with a resistance score of 1-3, and 35% of viruses with no predicted PI resistance. We correlated mutational prevalence to treatment duration in a treatment-characterized dataset of 2161 patients undergoing non-suppressive PI therapy. In the non-suppressed dataset, 24 mutations became more prevalent and three mutations became less prevalent after more than 48 months of non-suppressive PI-therapy. Longer durations of non-suppressive treatment correlated with higher PI resistance scores. Mutations at eight non-established positions that were more common in viruses with the longest duration of non-suppressive therapy were also more common in viruses with the highest PI resistance score. Covariation analysis of 3036 protease amino acid substitutions identified 75 positive and nine negative correlations between resistance associated positions. Our findings support the utility of reference laboratory datasets for surveillance of mutation prevalence and covariation.

Genome-wide analysis of chromosomal features repressing human immunodeficiency virus transcription.

We have investigated regulatory sequences in noncoding human DNA that are associated with repression of an integrated human immunodeficiency virus type 1 (HIV-1) promoter. HIV-1 integration results in the formation of precise and homogeneous junctions between viral and host DNA, but integration takes place at many locations. Thus, the variation in HIV-1 gene expression at different integration sites reports the activity of regulatory sequences at nearby chromosomal positions. Negative regulation of HIV transcription is of particular interest because of its association with maintaining HIV in a latent state in cells from infected patients. To identify chromosomal regulators of HIV transcription, we infected Jurkat T cells with an HIV-based vector transducing green fluorescent protein (GFP) and separated cells into populations containing well-expressed (GFP-positive) or poorly expressed (GFP-negative) proviruses. We then determined the chromosomal locations of the two classes by sequencing 971 junctions between viral and cellular DNA. Possible effects of endogenous cellular transcription were characterized by transcriptional profiling. Low-level GFP expression correlated with integration in (i) gene deserts, (ii) centromeric heterochromatin, and (iii) very highly expressed cellular genes. These data provide a genome-wide picture of chromosomal features that repress transcription and suggest models for transcriptional latency in cells from HIV-infected patients.

Small bowel obstruction--the water-soluble follow-through revisited.

AIMS: The aim of the present study to examine the use of a modified water-soluble follow-through in the diagnosis and management of small bowel obstruction (SBO). MATERIALS AND METHODS: Sixty-two patients were recruited to the study: 33 into the control group and 29 into the study group. A modified small bowel follow-through (SBFT) was performed in the study group patients. The control group was managed conventionally. Assessment was made by questionnaire documenting initial surgical diagnosis and likelihood of operative intervention, final diagnosis and surgical outcome (operative versus non-operative). RESULTS: SBFT changed the diagnosis in 12/24 of the study group (p<0.01). In the study group 8/24 proceeded to surgery whereas 19/33 underwent laparotomy in the control group, representing a relative risk reduction of 52%, but this was not statistically significant (0.10>p>0.05, chi-squared test). CONCLUSION: SBFT remains a valid and useful tool in surgical management of SBO. In particular it aids diagnostic confidence in planning surgical intervention, particularly in uncomplicated patients.

Sensitivity of multiplex real-time PCR reactions, using the LightCycler and the ABI PRISM 7700 Sequence Detection System, is dependent on the concentration of the DNA polymerase.

The introduction of multiplex PCR techniques to clinical laboratories has provided a means to streamline assays and to produce multiple results with minimal effort. While this methodology is very beneficial, care must be taken to ensure that reactions are properly optimized to allow for maximum sensitivity. This study was conducted to determine whether the sensitivity of multiplex-real-time PCR assays could be improved by increasing the concentration of DNA polymerase within a reaction. Multiplex reactions were designed to simultaneously detect the human HLA-DQ gene and a sequence from the UL83 region of the CMV genome. Two real-time PCR systems, one utilizing AmpliTaq Gold DNA polymerase and the ABI 7700 Sequence Detection System, and one utilizing FastStart Taq DNA polymerase and the Roche LightCycler were tested. The results indicated that increasing the AmpliTaq Gold concentration from 0.050 to 0.10 U/microl and the FastStart Taq concentration from 0.1875 to 0.375 U/microl increased detection sensitivity from 5,000 to 50 CMV copies per PCR reaction. In separate experiments, commercially prepared mastermixes were utilized for both real-time PCR platforms as per the manufacturer's suggestions or with the addition of supplemental DNA polymerase. In assays designed to detect 4 CMV genome copies per reaction, the addition of 2.5 U of AmpliTaq Gold to TaqMan Universal Mastermix increased the detection rate from 21 to 67%, and the addition of 5 U of FastStart Taq to FastStart DNA Master Hybridization Probes mastermix increased the detection rate from 17 to 56%. These results indicate that increasing the DNA polymerase concentration in multiplex real-time PCR reactions may be a simple way to optimize assay sensitivity.

A new guidance system for freehand, obstetric ultrasound-guided procedures.

OBJECTIVE: To assess the performance of the UltraGuide 1000 system, and to compare ultrasound-guided freehand mid-trimester amniocentesis with and without the new guidance system. METHODS: One hundred and sixty-nine women referred for mid-trimester genetic amniocentesis were divided into two groups: a control group of 99 women who underwent the procedure by the freehand technique with scored needles and 70 patients who had the procedure carried out with the aid of a guidance system (UltraGuide 1000) with non-scored needles. The procedures were compared for duration, number of punctures and repositionings of the needle, the visibility of the needle during the puncture and the number of bloody taps. RESULTS: The study group had significantly lower rates of reinsertion (none vs. 7.1%), repositioning (7.1% vs. 17.7%), bloody taps (none vs. 6.1%), touching the fetus (5.7% vs. 22.2%) and prolonged duration of the procedure (4.3% vs. 14.4%) compared with the control group. There was one fetal loss in the control group. Non-visibility of the needle before reaching the amniotic sac occurred in 18.6% of cases in the study group and in 38.4% of cases in the control group. CONCLUSIONS: The new guidance system combines the benefits of an attached guide with the flexibility of the 'freehand' technique. Use of the new guidance system for mid-trimester genetic amniocentesis increases needle visibility and lowers the incidence of common complications.

Immunization with Treponema pallidum outer membrane vesicles induces high-titer complement-dependent treponemicidal activity and aggregation of T. pallidum rare outer membrane proteins (TROMPs).

The purpose of this study was to determine whether immunization with purified outer membrane vesicles (OMV) from Treponema pallidum (T.p. ) could elicit Abs capable of killing this organism. It is well established that the immunization of rabbits or mice with killed T.p. or with recombinant T.p. Ags has failed to generate serum killing activity comparable with that of infection-derived immunity. Because of the small amount of T.p. OMV obtainable, a single mouse was immunized with purified OMV. The mouse anti-OMV serum and infection-derived immune rabbit serum (IRS) were compared by reactivities on two-dimensional T.p. immunoblots and by the T.p. immobilization test, a complement-dependent killing assay. Whereas IRS detected >40 Ags, the anti-OMV serum identified only 6 Ags corresponding to proteins identified previously in the outer membrane. T.p. immobilization testing showed that IRS had a 100% killing titer of 1:44 and a 50% killing titer of 1:662. By comparison, the mouse anti-OMV serum had a significantly greater 100% killing titer of 1:1,408 and a 50% killing titer of 1:16,896. Absorption of the anti-OMV serum to remove Ab against outer membrane-associated lipoproteins did not change the 100% killing titer. Freeze-fracture analysis of T.p. incubated in IRS or anti-OMV serum showed that T.p. rare membrane-spanning outer membrane proteins were aggregated. This is the first demonstration of high-titer killing Abs resulting from immunization with defined T.p. molecules; our study indicates that the targets for these Abs are T. p. rare outer membrane proteins.

Correlation of immunity in experimental syphilis with serum-mediated aggregation of Treponema pallidum rare outer membrane proteins.

We have previously shown by freeze-fracture electron microscopy that serum from infection-immune syphilitic rabbits aggregates the low-density membrane-spanning Treponema pallidum rare outer membrane proteins (TROMPs). The purpose of this study was to determine if a relationship could be demonstrated between acquired immunity in experimental rabbit syphilis, serum complement-dependent treponemicidal antibody, and antibody directed against TROMPs as measured by the aggregation of TROMP particles. Three groups of T. pallidum-infected rabbits were treated curatively with penicillin at 9 days, 30 days, and 6 months postinfection to generate various degrees of immunity to challenge reinfection. Sera from rabbits completely susceptible to localized and disseminated reinfection possessed a low titer of treponemicidal antibody (/=50% of a treponemal suspension) and showed a correspondingly low level of TROMP aggregation (16.5% of the total number of outer membrane particles counted) similar to normal serum controls (13. 4%); the number of particles within these aggregates never exceeded three. Sera from partially immune rabbits, which were susceptible to local reinfection but had no evidence of dissemination, showed an increase in the titer of treponemicidal antibody (1:16) compared to the completely susceptible group (

The outcome of contact procedures for tuberculosis in Edinburgh, Scotland 1982-1991.

We have reviewed the records of 632 (80%) of the 788 index cases of tuberculosis notified in Edinburgh from 1982-1991 to assess the value of contact procedures for tuberculosis. Screening was by tuberculin testing and radiological follow-up for 6 months. Fifty (7.9%) of 632 notifications were detected by contact procedures and a further 35 contacts had recent infection qualifying for chemoprophylaxis. Tuberculosis was diagnosed at the first clinic visit in 38 (76%) cases and a further 11 (22%) were diagnosed at 3 months. Twenty-seven (54%) contacts with tuberculosis were in the 0-14 year age group. BCG vaccination offered 59% protection. Forty-two (84%) cases of tuberculosis were in contacts of sputum smear-positive respiratory index cases. Contact procedures continue to be effective in identifying new cases of tuberculosis in Edinburgh. Most cases occur in children who are close contacts of smear-positive respiratory index cases and are identified within 3 months of initiating screening. Screening of close contacts other than those of smear-positive respiratory disease is usually unnecessary.

Treponemicidal antibody measured by the "washed-killing" assay correlates with immunity in experimental rabbit syphilis.

BACKGROUND AND OBJECTIVES: The authors have previously shown that complement-dependent treponemicidal antibody measured by the "washed-killing" assay is directed exclusively against surface-exposed targets on Treponema pallidum, presumably the Treponema pallidum rare outer membrane proteins detected by freeze-fracture electron microscopy. GOAL OF THIS STUDY: Because immune mechanisms against Treponema pallidum rare outer membrane proteins are likely to be central to a protective host response, it was examined whether a relationship could be established between treponemicidal levels as measured by the "washed-killing" assay and host immunity in experimental syphilis. STUDY DESIGN: Three groups of Treponema pallidum-infected rabbits were treated curatively with penicillin at 9 days, 30 days, and 6 months post-infection to generate animals with varying degrees of immunity to challenge re-infection. The level of complement-dependent treponemicidal activity in sera obtained before infection (basal) and before intradermal challenge was determined by the "washed-killing" assay and compared with that detected using conventional in vitro immobilization. RESULTS: Using the "washed-killing" assay, a close quantitative correlation as measured by a treponemal immobilizing endpoint titer was demonstrable between prechallenge treponemicidal antibody and the status of immunity to re-infection. Sera from rabbits completely susceptible to symptomatic and disseminated asymptomatic re-infection lacked treponemicidal antibody. Sera from challenged rabbits with a relatively low degree of immunity to symptomatic disease showed endpoints of < or = 4. Rabbits with a relatively high degree of immunity to symptomatic reinfection and resistant to disseminated disease had endpoints that ranged from 6 to 96. Rabbits completely resistant to challenge exhibited endpoints ranging from 96 to 128. CONCLUSION: Treponemicidal antibody measured by the "washed-killing" assay correlated closely with the status of immunity in experimental rabbit syphilis. Thus, antibody measured by this assay may be directed against key protective Treponema pallidum surface immunogens.

Intracellular free magnesium in lymphocytes from patients with congestive cardiac failure treated with loop diuretics with and without amiloride.

Cellular Mg2+ depletion has been reported in patients on long term diuretic therapy. We therefore measured lymphocyte free Mg2+ concentrations in patients with congestive cardiac failure treated with loop diuretics or a frusemide/amiloride mixture (Frumil) and compared them with control subjects. There was no correlation between lymphocyte free Mg2+ levels and plasma Mg2+ concentrations. Patients treated with loop diuretics did not show lymphocyte intracellular free Mg2+ depletion compared with normal controls, and those on Frumil showed higher intracellular free Mg2+ levels than normal.