A diagnostic approach to confirm Mycoplasma hyopneumoniae "Day zero" for pathogen eradication.

Breeding herds in the US are trending toward eradication of Mycoplasma hyopneumoniae (M. hyopneumoniae) due to the positive impact on welfare and downstream production. In an eradication program, "Day 0″ is the time point when the last replacement gilts to enter the herd were exposed to M. hyopneumoniae and marks the beginning of a herd closure. However, no specific diagnostic protocols are available for confirmation of successful exposure to define Day 0. Therefore, the objective of this study was to develop diagnostic guidelines, including sample collection approaches, for two common gilt exposure methods to confirm an entire population has been infected with M. hyopneumoniae following purposeful exposure. Forty gilts, age 21-56 days, were ear-tagged for longitudinal sample collection at five commercial gilt developer units (GDUs) and were exposed to M. hyopneumoniae by natural contact or aerosolization. Study gilts originated from sources known to be negative to major swine pathogens, including M. hyopneumoniae, and were sampled prior to exposure to confirm negative status, then every two weeks. Blood samples were collected for antibody detection, while laryngeal and deep tracheal secretions and pen based oral fluids were collected for PCR, and sampling continued until at least 85% of samples were positive by PCR. Detection of M. hyopneumoniae varied greatly based on sample type. Oral fluids showed the lowest detection in groups of gilts detected positive by other sample types. Detection by PCR in deep tracheal secretions was higher than in laryngeal secretions. Seroconversion to and PCR detection of M. hyopneumoniae on oral fluids were delayed compared to PCR detection at the individual level. By two weeks post-exposure, at least 85% of study gilts in three GDUs exposed by aerosolization were PCR positive in deep tracheal secretions. Natural contact exposure resulted in 22.5% of study gilts becoming PCR positive by two weeks post-initial exposure, 61.5% at four weeks, and 100% at six weeks on deep tracheal secretions. Deep tracheal secretions required the lowest number of gilts to sample for the earliest detection compared to all other samples evaluated. As observed in one of the GDUs using aerosolization, demonstration of failure to expose gilts to M. hyopneumoniae allowed for early intervention in the exposure protocol and delay of Day 0, for accurate timing of the eradication protocol. Sampling guidelines proposed in this study can be used for verification of M. hyopneumoniae infection of gilts following exposure to determine Day 0 of herd closure.

Duration of Mycoplasma hyopneumoniae detection in pigs following purposeful aerosol exposure.

Swine disease elimination programs for Mycoplasma hyopneumoniae are commonly applied in the North American swine industry and may include the aerosolization of medium containing lung tissue to achieve population exposure prior to start. Field data has indicated M. hyopneumoniae PCR detection in pigs beyond 240 days post-herd closure (dphc; planned end of an elimination program) and is thought to contribute to disease elimination programs' failure. Here, the duration of M. hyopneumoniae detection in sows and replacement gilts following aerosolized lung homogenate exposure, as part of a dual disease elimination program, was determined. A subset of sows and gilts from a commercial sow herd and off-site gilt development unit were longitudinally sampled to collect deep tracheal catheter secretions at various times post-exposure. Samples were tested for M. hyopneumoniae using a species-specific real-time PCR. A proportion of 58, 51, 52, 19, and 2% females were detected positive at 30, 60, 120, 180 and 240 dphc, respectively. Noteworthy, a greater proportion of gilts exposed at the off-site GDU were detected PCR positive for M. hyopneumoniae at each sampling event, compared to sows. In this study, assaying for genetic material in live female pigs showed extended detection of M. hyopneumoniae until at least 240 dphc. This data suggests persistence of M. hyopneumoniae longer than previously reported and highlights the importance of performing diagnostic testing to confirm negativity to the bacterium, prior to opening sow herds, especially late in the herd closure timeline.

Evaluation of dam parity and internal biosecurity practices in influenza infections in piglets prior to weaning.

Influenza is an important respiratory disease of pigs and humans. Controlling influenza in pigs is challenging due to the substantial genetic diversity of influenza A virus (IAV). In this study, we assessed the impact of internal biosecurity practices directed at limiting exposure of piglets to IAV before weaning; evaluated the association of sow parity with IAV prevalence in piglets and the levels of maternally derived antibodies (MDA), and documented the frequency of detection of IAV on farmworkers' hands and the instruments used when handling pigs. The control group included litters in rooms where no specific changes were made to standard farm procedures. The treatment group included litters in rooms where no cross-fostering or nurse sows use was allowed, and where farmworkers were required to change gloves between litters when handling pigs. Both, younger (≤ Parity 3) and older parity sows (>Parity 3) were represented in all rooms included in the study. Overall, litters in the treatment group had lower IAV prevalence (29.9 %) than litters in the control group (44.2 %) (p < 0.001), and at day 8 of age the litters from the control group had 7.5 times higher IAV prevalence than the litters from the treatment group. However, at weaning differences were not found (77.2 % vs. 81 % for treatment vs. control, respectively, p = 0.41). There were no differences in IAV detection between parity groups at any of the sampling points (p = 0.86) and incidence of detection in sows from farrowing to weaning was 29 %. Piglets that tested ELISA negative were 1.3 times more likely to test IAV positive than piglets that were ELISA positive for IAV antibody test, suggesting that effective colostrum intake may reduce the likelihood of infection. IAV was detected on 46 % of the instruments used when handling piglets and on 58 % of farmworkers' hands, indicating the potential risk for mechanical transmission of IAV in pigs. Overall, we showed that the implementation of internal biosecurity practices that limit IAV exposure to newborn piglets helped delay IAV infections but were not sufficient to reduce the prevalence of IAV infection in litters at weaning.

A novel disinfection protocol using ATP testing for lead garments in the electrophysiology lab.

PURPOSE: Electrophysiology procedures pose infection risk and require surgical room sterility. Currently, there is no universally approved protocol for disinfecting lead garments in the electrophysiology laboratory. This study explores the feasibility of using ATP testing to assess the microbial burden of lead aprons and evaluates the impact of a sanitary intervention. METHODS: Adenosine triphosphate (ATP) testing is a well-established hospital standard to quantify biological matter on a surface and, by proxy, the microbial burden. It is measured in RLU (relative light units). Pre-intervention ATP testing was performed on 34 lead garments after use for electrophysiology procedures. The thyroid collar, mid-chest vest, and left axillary areas of the garments were swabbed using a Hygiena SystemSure II luminometer with ATP swabs (Hygiena, Camarillo, CA). These sites were then disinfected with disinfectant wipes (PDI Super Sani-cloth Germicidal Disposable Wipe) and ATP testing was repeated. RESULTS: The mean duration of garment wear was 213 min. The thyroid collars had the highest mean RLU before intervention, followed by the mid-chest vest and the left axillary areas. The intervention was found to significantly decrease ATP readings for all three sites (p = 0.0002, p = 0.0001, p = 0.0002 respectively). Linear regression modeling to assess the impact of intervention showed a significant correlation with pre-intervention ATP values for all three sites but no correlation with fluoroscopy time, fluoroscopy dose, or total time spent within the procedure. CONCLUSIONS: Lead garments harbor microbial contamination after use according to ATP testing. A sanitary intervention can decontaminate lead garments and potentially reduce rates of hospital infection.

Unified Extracurricular Activities as a Pathway to Social Inclusion in High Schools.

The present study examined how a multicomponent intervention embedded in a high school's extracurricular framework impacts students' acceptance of peers with intellectual disability (ID). Data were collected from eight high schools, three of which implemented the Special Olympics Unified Champion Schools (UCS) program involving inclusive sports, clubs, and schoolwide events, and five of which did not. A pretest-posttest survey design was used to measure students' attitudes, perceptions, and interactions (n = 1,230). Lagged dependent variable modeling revealed that UCS participation significantly predicted improved attitudes toward peers with ID and perceptions of school social inclusion, as well as increased social interactions with peers with ID. Unified extracurricular activities may be the next step forward in promoting an inclusive school culture.

Impact of Flow Rate, Collection Devices, and Extraction Methods on Tear Concentrations Following Oral Administration of Doxycycline in Dogs and Cats.

PURPOSE: Compare the precision of doxycycline quantification in tear fluid collected with either Schirmer strips or polyvinyl acetal (PVA) sponges following oral drug administration. METHODS: Three dogs and 3 cats were administered doxycycline orally at a dose of 4.2-5 mg/kg every 12 h for 6 consecutive days. At day 5 and 6, blood and tear fluid were sampled to capture doxycycline trough and maximal concentrations. Tear fluid was collected 3 times (spaced 10 min apart) at each session with the absorbent material placed in the lower conjunctival fornix until the 20-mm mark was reached (Schirmer strip, one eye) or for 1 min (PVA sponge, other eye). Tear extraction was performed with either centrifugation or elution in methanol. Doxycycline concentrations were measured with liquid chromatography-mass spectrometry. Low (100 ng/mL) and high (1,000 ng/mL) tear concentrations measured in vivo were spiked into each absorbent material in vitro to evaluate percentage drug recovery. RESULTS: After oral administration of doxycycline, the drug reached the tear compartment at concentrations of 45.1-900.7 ng/mL in cats and 45.4-632.0 ng/mL in dogs, representing a tear-to-serum ratio of 12% and 16%, respectively. Doxycycline tear concentrations were significantly more precise when tear collection was performed with Schirmer strips rather than PVA sponges (P = 0.007), but were not correlated with tear flow rate. In vitro doxycycline recovery was poor to moderate (<75%). CONCLUSIONS: Schirmer strips represent a good option for lacrimal doxycycline quantification, although the collection and subsequent extraction have to be optimized to improve drug recovery.

Effect of tear collection on lacrimal total protein content in dogs and cats: a comparison between Schirmer strips and ophthalmic sponges.

BACKGROUND: Quantification of lacrimal total protein content (TPC) is an important tool for clinical scientists to understand disease pathogenesis, identify potential biomarkers and assess response to therapy, among other applications. However, TPC is not only affected by disease state but also by the method used for tear collection. Thus, the purpose of this study is to determine the impact on TPC of two methods of tear collection in dogs and cats: Schirmer strips and polyvinyl acetal (PVA) sponges. METHODS: (i) In vivo - Ten healthy dogs and 10 healthy cats were examined. Each animal underwent two sessions, separated by 10 min, in which a Schirmer strip was placed in one randomly selected eye until the 20-mm mark was reached, while a strip of PVA sponge was placed in the other eye for 1 min. (ii) In vitro - Schirmer strips and PVA sponges were spiked with various volumes of four bovine serum albumin solutions (0.5, 4, 10, and 20 mg/mL). In both experiments, the wetted absorbent materials were centrifuged for 1 min, and the TPC was quantified on the extracted fluid using Direct Detect™ infrared spectroscopy. RESULTS: Lacrimal TPC in dogs and cats ranged from 5.2 to 14.6 mg/mL and from 6.2 to 20.6 mg/mL, respectively. In cats, TPC was significantly lower with Schirmer strips vs. PVA sponges (P < 0.001). In dogs, the volume absorbed by PVA sponges was negatively correlated with TPC (r = - 0.48, P = 0.033). The inter-session coefficient of variation was significantly lower with Schirmer strips vs. PVA sponges in both species (P ≤ 0.010). In vitro, both absorbent materials resulted in a 'concentrating effect' of the TPC obtained post-centrifugation, which was most pronounced when the volume absorbed was low, especially for Schirmer strips. CONCLUSION: Schirmer strips provide a repeatable method to quantify lacrimal TPC in dogs and cats, although care should be taken to absorb sufficient volumes of tears to minimize the concentrating effect from the absorbent material.

Cardiorenal syndrome: understanding the connections between cardiac and renal disease.

Renal and cardiac diseases are nearly ubiquitous in hospitalized patients and common causes of morbidity in outpatients. Although the connection between the heart and kidneys is relatively well known in the medical community, a more formal classification for the clinical interplay of the two systems has been developed only recently. Cardiorenal syndrome was described by Italian nephrologist Claudio Ronco in 2008. This classification allows for justification of management strategies in these complex patients and will guide further research studies.

Hippocampal volumes in patients with chronic combat-related posttraumatic stress disorder: a systematic review.

The authors and others have recently demonstrated that veterans with chronic combat-related PTSD (CR-PTSD) have a twofold increased risk of dementia. To understand this increased incidence, they performed a systematic review of the literature on neuroanatomical differences between veterans with chronic CR-PTSD and control subjects (22 included studies). The hippocampus was most commonly and consistently reported to differ between groups, thereby suggesting the hypothesis that PTSD is associated with smaller hippocampi, which increases the risk for dementia. However, an alternate hypothesis is that smaller hippocampal volumes are a preexisting risk factor for PTSD and dementia. Studies are clearly needed to differentiate between these important possibilities.

CodY-mediated regulation of Streptococcus pyogenes exoproteins.

BACKGROUND: The production of Streptococcus pyogenes exoproteins, many of which contribute to virulence, is regulated in response to nutrient availability. CodY is a transcriptional regulator that controls gene expression in response to amino acid availability. The purpose of this study was to identify differences in the expression of streptococcal exoproteins associated with deletion of the codY gene. RESULTS: We compared the secreted proteins produced by wild-type S. pyogenes to a codY mutant in the post-exponential phase of growth. We used both one and two-dimensional gel electrophoresis to separate exoproteins. Proteins that were significantly different in abundance upon repeated analysis were identified with tandem mass spectrometry. The production of the secreted cysteine protease SpeB, a secreted chromosomally encoded nuclease (SdaB), and a putative adhesion factor (Spy49_0549) were more abundant in supernatant fluids obtained from the codY mutant. In addition, hyaluronidase (HylA), CAMP factor (Cfa), a prophage encoded nuclease (Spd-3), and an uncharacterized extracellular protein (Spy49_0015) were less abundant in supernatant fluids obtained from the codY mutant strain. Enzymatic assays showed greater DNase activity in culture supernatants isolated in the post-exponential phase of growth from the codY mutant strain compared to the wild-type strain. Because extracellular nucleases and proteases can influence biofilm formation, we also measured the ability of the strains to form biofilms during growth with both rich medium (Todd Hewitt yeast extract; THY) and chemically defined media (CDM). No difference was observed with rich media but with CDM the biofilms formed by the codY mutant strain had less biomass compared to the wild-type strain. CONCLUSIONS: Overall, the results indicate that CodY alters the abundance of a select group of S. pyogenes exoproteins, including DNases, a protease, and hylauronidase, which together may alleviate starvation by promoting dissemination of the pathogen to nutrient rich environments and by hydrolysis of host macromolecules.

Physiological levels of ATP negatively regulate proteasome function.

Intracellular protein degradation by the ubiquitin-proteasome system is ATP dependent, and the optimal ATP concentration to activate proteasome function in vitro is ∼100 µM. Intracellular ATP levels are generally in the low millimolar range, but ATP at a level within this range was shown to inhibit proteasome peptidase activities in vitro. Here, we report new evidence that supports a hypothesis that intracellular ATP at the physiological levels bidirectionally regulates 26S proteasome proteolytic function in the cell. First, we confirmed that ATP exerted bidirectional regulation on the 26S proteasome in vitro, with the optimal ATP concentration (between 50 and 100 µM) stimulating proteasome chymotrypsin-like activities. Second, we found that manipulating intracellular ATP levels also led to bidirectional changes in the levels of proteasome-specific protein substrates in cultured cells. Finally, measures to increase intracellular ATP enhanced, while decreasing intracellular ATP attenuated the ability of proteasome inhibition to induce cell death. These data strongly suggest that endogenous ATP within the physiological concentration range can exert a negative impact on proteasome activities, allowing the cell to rapidly upregulate proteasome activity on ATP reduction under stress conditions.

Community-based intervention to manage an outbreak of MRSA skin infections in a county jail.

This article describes a community-based intervention to manage an outbreak of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) skin infections in a midwestern county jail. A systematic investigation conducted by a family medicine residency program identified 64 total cases and 19 MRSA cases between January 1 and December 31, 2007. Factors contributing to MRSA transmission included inadequate surveillance, lack of antibacterial soap, and a defective laundry process. All 19 isolates were CA-MRSA and all seven tested by pulsed-field gel electrophoresis (PFGE) were USA300. Four of the seven isolates showed variation of their PFGE patterns. A primary care approach using community-based resources effectively reduced the number of cases in this heterogeneous outbreak of CA-MRSA, with the last MRSA being isolated in October 2007.

Turbidity suppression from the ballistic to the diffusive regime in biological tissues using optical phase conjugation.

We describe the amplitude and resolution trends of the signals acquired by turbidity suppression through optical phase conjugation (TSOPC) with samples that span the ballistic and diffusive scattering regimes. In these experiments, the light field scattered through a turbid material is written into a hologram, and a time-reversed copy of the light field is played back through the sample. In this manner, the wavefront originally incident on the sample is reconstructed. We examine a range of scattering samples including chicken breast tissue sections of increasing thickness and polyacrylamide tissue-mimicking phantoms with increasing scattering coefficients. Our results indicate that only a small portion of the scattered wavefront (<0.02%) must be collected to reconstruct a TSOPC signal. Provided the sample is highly scattering, all essential angular information is contained within such small portions of the scattered wavefront due to randomization by scattering. A model is fitted to our results, describing the dependence of the TSOPC signal on other measurable values within the system and shedding light on the efficiency of the phase conjugation process. Our results describe the highest level of scattering that has been phase conjugated in biological tissues to date.

An in vivo study of turbidity suppression by optical phase conjugation (TSOPC) on rabbit ear.

We present a holography-based in vivo optical phase conjugation experiment performed on a living rabbit ear. The motion of live tissues caused the phase conjugate signal to decay with a consistent decay time of less than two seconds. We monitor the signal decay time variation after the ear is excised to postulate different mechanisms that cause the signal decay. The experimental findings address the minimum speed limit of a broad range of optical time reversal experiments for in vivo applications on tissues.

Association between dysarthria and cognitive impairment in ALS: A prospective study.

Several studies have demonstrated impaired cognition in amyotrophic lateral sclerosis (ALS) patients, but it has been difficult to identify risk factors for this impairment. An association between cognitive changes and bulbar site of onset or dysarthria has been suggested, but the findings are variable. We tested for both associations in a large cohort of ALS patients. At the time of diagnosis of sporadic ALS, all patients (n=355) in this prospective study underwent comprehensive neuropsychological testing. In addition, a subset of 175 patients underwent a detailed assessment of dysarthria, which was quantified using the Appel ALS Score (AALSS). ALS patients with bulbar site of onset performed significantly worse than limb onset patients on a few timed ((VSAT-time, p<0.05), (Stroop Color, p<0.05), (Stroop Word, p<0.05)) tests of frontal lobe functions, but the significance could not be replicated when motor impairment was accommodated into the tests ((VSAT-errors, p=0.73), (Stroop interference, p=0.08)). ALS patients with dysarthria performed significantly worse than non-dysarthrics on multiple timed ((BD, p<0.05), (VSAT-time, p<0.05), (Stroop Color, p<0.05), (Stroop Word, p<0.05), (Trails A, p<0.05), (Trails B, p<0.05)) neuropsychological tests, and the significance was maintained when motor impairment was accommodated into one of these tests (Stroop interference, p<0.05). Additionally, dysarthrics performed significantly worse on two untimed measures of cognition ((Similarities, p<0.05), (Rey Copy, p<0.05)). Cognitive functioning in ALS does not associate with the site of onset and has a moderate association with dysarthria.

Manual-scanning optical coherence tomography probe based on position tracking.

A method based on position tracking to reconstruct images for a manual-scanning optical coherence tomography (OCT) probe is proposed and implemented. The method employs several feature points on a hand-held probe and a camera to track the device's pose. The continuous device poses tracking, and the collected OCT depth scans can then be combined to render OCT images. The tracking accuracy of the system was characterized to be about 6 microm along two axes and 19 microm along the third. A phantom target was used to validate the method. In addition, we report OCT images of a 54-stage Xenopus laevis tadpole acquired by manual scanning.

Observation of polarization-gate based reconstruction quality improvement during the process of turbidity suppression by optical phase conjugation.

We present experiments that study the impact of polarization selection on the phenomenon of turbidity suppression by optical phase conjugation. Counter to intuition, we discovered that the preferential utilization of multiply scattered light field components over their sparsely scattered counterparts via appropriate polarization selection can lead to better image reconstruction quality. This effect was observed with tissue phantoms and biological tissue sections. The physical origin of this effect and its dependence on scatterer properties are discussed.

A naturally occurring Rgg variant in serotype M3 Streptococcus pyogenes does not activate speB expression due to altered specificity of DNA binding.

The transcriptional regulator Rgg of Streptococcus pyogenes is essential for expression of the secreted cysteine protease SpeB. Although all isolates of S. pyogenes possess the speB gene, not all of them produce the protein in vitro. In a murine model of infection, the absence of SpeB production is associated with invasive disease. We speculated that naturally occurring mutations in rgg, which would also abrogate SpeB production, may be present in invasive isolates of S. pyogenes. Examination of the inferred Rgg sequences available in public databases revealed that the rgg gene in strain MGAS315 (a serotype M3 strain associated with invasive disease) encodes a proline at amino acid position 103 (Rgg(103P)); in contrast, all other strains encode a serine at this position (Rgg(103S)). A caseinolytic assay and Western blotting indicated that strain MGAS315 does not produce SpeB in vitro. Gene-swapping experiments showed that the rgg gene of MGAS315 is solely responsible for the lack of SpeB expression. In contrast to Rgg(103S), Rgg(103P) does not bind to the speB promoter in gel shift assays, which correlates with a lack of speB expression. Despite its inability to activate speB expression, Rgg(103P) retains the ability to bind to DNA upstream of norA and to influence its expression. Overall, this study illustrates how variation at the rgg locus may contribute to the phenotypic diversity of S. pyogenes.

Fundamental sensitivity limit imposed by dark 1/f noise in the low optical signal detection regime.

The impact of dark 1/f noise on fundamental signal sensitivity in direct low optical signal detection is an understudied issue. In this theoretical manuscript, we study the limitations of an idealized detector with a combination of white noise and 1/f noise, operating in detector dark noise limited mode. In contrast to white noise limited detection schemes, for which there is no fundamental minimum signal sensitivity limit, we find that the 1/f noise characteristics, including the noise exponent factor and the relative amplitudes of white and 1/f noise, set a fundamental limit on the minimum signal that such a detector can detect.

Inter- and intraserotypic variation in the Streptococcus pyogenes Rgg regulon.

Human isolates of Streptococcus pyogenes, a Gram-positive bacterium, are characterized by significant genetic and phenotypic variation. The rgg locus, also known as ropB, is a global transcriptional regulator of genes associated with metabolism, stress responses, and virulence in S. pyogenes strain NZ131 (serotype M49). To assess the breadth of the Rgg regulon, the rgg gene was inactivated in three additional strains representing serotypes M1 (strains SF370 and MGAS5005) and M49 (strain CS101). Changes in gene expression were identified in the postexponential phase of growth using Affymetrix NimbleExpress Arrays. The results identified an Rgg core-regulon consisting of speB and adjacent hypothetical protein gene, spy2040, and a variable and strain-specific subregulon, ranging in size from a single gene (spy1793) in strain MGAS5005 to 43 genes in strain SF370. Thus, both interserotypic and intraserotypic variation is characteristic of the Rgg regulon in S. pyogenes.