Understanding the Molecular Epidemiology of the Footrot Pathogen Dichelobacter nodosus To Support Control and Eradication Programs

The Gram-negative anaerobe Dichelobacter nodosus is the primary etiologic agent of ovine footrot. Few studies of the genetic diversity and epidemiology of D. nodosus have been done, despite the economic cost and welfare implications of the disease. This study examined a large collection of Australian isolates; 735 isolates from footrot-infected sheep from 247 farms in Western Australia (WA) were tested by pulsed-field gel electrophoresis (PFGE), and a subset of 616 isolates was tested by infrequent restriction site PCR (IRS-PCR). The genetic diversity of WA isolates was compared to that of 61 isolates from three other Australian states. WA isolates were genetically diverse, with 181 molecular types resolved by PFGE, resulting in a simple diversity ratio (SDR) of 1:4 and a Simpson''s index of discrimination value (D) of 0.98. IRS-PCR resolved 77 molecular types (SDR = 1:8 and D = 0.95). The isolates were grouped into 67 clonal groups by PFGE (SDR = 1:11, D = 0.90) and 36 clonal groups by IRS-PCR (SDR = 1:17, D = 0.87). Despite the high genetic diversity, three common clonal groups predominated in WA and were found in other Australian states. On some farms, molecular type was stable over a number of years, whereas on other farms genetically diverse isolates occurred within a flock of sheep or within a hoof. This study provides a large database from which to appropriately interpret molecular types found in epidemiological investigations and to identify common and unknown types that may compromise footrot eradication or control programs.Footrot is a contagious disease affecting the hooves of sheep, goats, and occasionally cattle, with the primary etiological agent being the anaerobic bacterium Dichelobacter nodosus (2). The bacterium is an obligate parasite of the hoof and survives for a limited time outside the host.In Western Australia (WA), a footrot eradication program existed between 1974 and 2006 until a control program was implemented in 2007. Under the programs, infected sheep are identified, quarantined, and treated (8). Understanding the risk factors for disease transmission improves the effectiveness of eradication or control programs. Risk factors include transmissibility of the organism, environmental factors, phenotype, virulence potential of different strains, or as in the case of footrot, clinically expressed virulence that is dependent upon optimal environmental conditions (4). Molecular epidemiology can provide information about the risk factors associated with different bacterial strains—associations between particular genotypes and phenotypes, including strain virulence and transmissibility, interstrain genetic relatedness, and identification of clonal groups—and adds to information gathered for trace-back of infection outbreaks (14). An effective molecular typing system identifies genetic diversity such that organisms isolated at different times from different geographical locations and from different hosts can be differentiated or classified into subtypes of strains (15). The pulsed-field gel electrophoresis (PFGE) method is considered the “gold standard” for molecular epidemiological studies of bacteria because it is reliable, reproducible, highly discriminatory, and sensitive enough to detect recent evolutionary divergence that may occur during a disease outbreak (1). PFGE was used on D. nodosus in a small study of three farms in Malaysia, in which 12 isolates were differentiated into eight molecular types (24), suggesting considerable diversity, despite the small number of isolates tested.The purpose of this study was to use two molecular typing methods (PFGE and infrequent restriction site PCR [IRS-PCR]) to study a large collection of D. nodosus isolates and to determine the suitability of these methods for investigation of the molecular epidemiology of D. nodosus during infection outbreaks and over the long term. It was anticipated that these methods would assist in ongoing eradication and control programs for ovine footrot in WA.     

A role for direct interactions in the modulation of rhodopsin by omega-3 polyunsaturated lipids

Rhodopsin, the G protein-coupled receptor primarily responsible for sensing light, is found in an environment rich in polyunsaturated lipid chains and cholesterol. Biophysical experiments have shown that lipid unsaturation and cholesterol both have significant effects on rhodopsin's stability and function; omega-3 polyunsaturated chains, such as docosahexaenoic acid (DHA), destabilize rhodopsin and enhance the kinetics of the photocycle, whereas cholesterol has the opposite effect. Here, we use molecular dynamics simulations to investigate the possibility that polyunsaturated chains modulate rhodopsin stability and kinetics via specific direct interactions. By analyzing the results of 26 independent 100-ns simulations of dark-adapted rhodopsin, we found that DHA routinely forms tight associations with the protein in a small number of specific locations qualitatively different from the nonspecific interactions made by saturated chains and cholesterol. Furthermore, the presence of tightly packed DHA molecules tends to weaken the interhelical packing. These results are consistent with recent NMR work, which proposes that rhodopsin binds DHA, and they suggest a molecular rationale for DHA's effects on rhodopsin stability and kinetics.     

Inflammatory foreign-body reaction to an arthroscopic bioabsorbable meniscal arrow repair.

Various arthroscopic meniscal repair techniques have been developed in recent years to preserve meniscal function. We report the case of a patient with a failed arthroscopic meniscal repair demonstrating an inflammatory foreign-body reaction to bioabsorbable meniscal arrows.     

Automated algorithm for differentiation of human breast tissue using low coherence interferometry for fine needle aspiration biopsy guidance

Fine needle aspiration biopsy (FNAB) is a rapid and cost-effective method for obtaining a first-line diagnosis of a palpable mass of the breast. However, because it can be difficult to manually discriminate between adipose tissue and the fibroglandular tissue more likely to harbor disease, this technique is plagued by a high number of nondiagnostic tissue draws. We have developed a portable, low coherence interferometry (LCI) instrument for FNAB guidance to combat this problem. The device contains an optical fiber probe inserted within the bore of the fine gauge needle and is capable of obtaining tissue structural information with a spatial resolution of 10 mum over a depth of approximately 1.0 mm. For such a device to be effective clinically, algorithms that use the LCI data must be developed for classifying different tissue types. We present an automated algorithm for differentiating adipose tissue from fibroglandular human breast tissue based on three parameters computed from the LCI signal (slope, standard deviation, spatial frequency content). A total of 260 breast tissue samples from 58 patients were collected from excised surgical specimens. A training set (N=72) was used to extract parameters for each tissue type and the parameters were fit to a multivariate normal density. The model was applied to a validation set (N=86) using likelihood ratios to classify groups. The overall accuracy of the model was 91.9% (84.0 to 96.7) with 98.1% (89.7 to 99.9) sensitivity and 82.4% (65.5 to 93.2) specificity where the numbers in parentheses represent the 95% confidence intervals. These results suggest that LCI can be used to determine tissue type and guide FNAB of the breast.     

Genome,Proteome and Structure of a T7-Like Bacteriophage of the Kiwifruit Canker Phytopathogen Pseudomonas syringae pv. actinidiae

Pseudomonas syringae pv. actinidiae is an economically significant pathogen responsible for severe bacterial canker of kiwifruit (Actinidia sp.). Bacteriophages infecting this phytopathogen have potential as biocontrol agents as part of an integrated approach to the management of bacterial canker, and for use as molecular tools to study this bacterium. A variety of bacteriophages were previously isolated that infect P. syringae pv. actinidiae, and their basic properties were characterized to provide a framework for formulation of these phages as biocontrol agents. Here, we have examined in more detail φPsa17, a phage with the capacity to infect a broad range of P. syringae pv. actinidiae strains and the only member of the Podoviridae in this collection. Particle morphology was visualized using cryo-electron microscopy, the genome was sequenced, and its structural proteins were analysed using shotgun proteomics. These studies demonstrated that φPsa17 has a 40,525 bp genome, is a member of the T7likevirus genus and is closely related to the pseudomonad phages φPSA2 and gh-1. Eleven structural proteins (one scaffolding) were detected by proteomics and φPsa17 has a capsid of approximately 60 nm in diameter. No genes indicative of a lysogenic lifecycle were identified, suggesting the phage is obligately lytic. These features indicate that φPsa17 may be suitable for formulation as a biocontrol agent of P. syringae pv. actinidiae.     

Patient‐Reported Symptom Control of Diarrhea and Flushing in Patients with Neuroendocrine Tumors Treated with Lanreotide Depot/Autogel: Results from a Randomized,Placebo‐Controlled,Double‐Blind and 32‐Week Open‐Label Study

Background

In the double‐blind (DB) ELECT study, lanreotide depot/autogel significantly reduced versus placebo the need for short‐acting octreotide for symptomatic carcinoid syndrome (CS) control in neuroendocrine tumor (NET) patients. Here we present patient‐reported symptom data during DB and initial open‐label (IOL) treatment.

Materials and Methods

Adults with NETs and CS history, with/without prior somatostatin analog use, were randomized to 16 weeks’ DB lanreotide 120 mg subcutaneous or placebo every 4 weeks, followed by 32 weeks’ IOL lanreotide. Patients recorded diarrhea and/or flushing frequency and severity daily by Interactive Voice (Web) Response System for 1 month prior to randomization and throughout the study.

Results

Of 115 patients randomized (n = 59 lanreotide, n = 56 placebo), 56 lanreotide and 45 placebo patients enrolled in the IOL phase. During DB treatment, least square (LS) mean percentages of days with moderate/severe diarrhea and/or flushing were significantly lower for lanreotide (23.4%) versus placebo (35.8%; LS mean difference [95% confidence interval]: ?12.4 [?20.73 to ?4.07]; p = .004). For DB lanreotide patients, average daily composite (frequency × severity) diarrhea scores improved significantly between DB and IOL treatment (mean difference: ?0.71 [?1.20 to ?0.22]; p = .005), and remained stable for diarrhea and/or flushing. For DB placebo patients, composite scores for diarrhea, flushing, and diarrhea and/or flushing improved significantly between DB and IOL treatment (mean differences: ?1.07 [?1.65 to ?0.49]; ?1.06 [?1.93 to ?0.19]; and ?2.13 [?3.35 to ?0.91]; all p ≤ .018).

Conclusion

Improved diarrhea and flushing control in CS patients during 16‐week lanreotide treatment was sustained during maintenance of lanreotide treatment for the 32‐week IOL phase (48 weeks total).

Implications for Practice

This study prospectively collected daily patient‐reported data on diarrhea and flushing from the ELECT trial to evaluate the direct impact of lanreotide depot on patients’ relief of carcinoid syndrome symptoms. Treatment with lanreotide depot was associated with significant reductions in the percentages of days patients reported symptoms of diarrhea and flushing, as well as reductions in the frequency and severity of daily symptoms compared with placebo during 16 weeks of double‐blind treatment. These improvements were sustained for 32 additional weeks of open‐label lanreotide treatment (i.e., through week 48 of treatment), resulting in clinically meaningful, long‐term symptom reduction.     

Effect of stressor intensity on habituation of the adrenocortical stress response

Although it is known that the number of presentations of a stressor can influence the adrenocortical stress response, relatively little information exists on how stressor intensity affects this process. To evaluate this, we repeatedly presented rats with stressors of 3 different intensities and sampled blood for corticosterone. The first major finding was that the rat's initial adrenocortical responsiveness regardless of the stressor employed was a critical variable. Rats that showed a small corticosterone response showed no evidence of habituation or of differences due to stressor intensity. Rats that showed an initial robust response all showed partial habituation of their corticosterone response over time but the patterns varied with stressor intensity. Handled and prone restrained rats showed the same pattern but rats subjected to the more intense stressor of supine restraint showed delay in habituation and tonically elevated responses. These data indicate that individual differences in reactivity to stressors as well as stressor intensity can influence the pattern of the stress response over the course of repeated administration of the stressor.