Cooled radiofrequency ablation provides extended clinical utility in the management of knee osteoarthritis: 12-month results from a prospective, multi-center, randomized, cross-over trial comparing cooled radiofrequency ablation to a single hyaluronic acid injection.

BACKGROUND: Safe and effective non-surgical treatments are an important part of the knee osteoarthritis (OA) treatment algorithm. Cooled radiofrequency ablation (CRFA) and hyaluronic acid (HA) injections are two commonly used modalities to manage symptoms associated with knee OA. METHODS: A prospective 1:1 randomized study was conducted in 177 patients comparing CRFA to HA injection with follow-ups at 1, 3, 6 and 12 months. HA subjects with unsatisfactory outcomes at 6-months were allowed to crossover and receive CRFA. Knee pain (numeric rating scale = NRS), WOMAC Index (pain, stiffness and physical function), overall quality of life (global perceived effect = GPE, EQ-5D-5 L), and adverse events were measured. RESULTS: At 12-months, 65.2% of subjects in the CRFA cohort reported ≥50% pain relief from baseline. Mean NRS pain score was 2.8 ± 2.4 at 12 months (baseline 6.9 ± 0.8). Subjects in the CRFA cohort saw a 46.2% improvement in total WOMAC score at the 12-month timepoint. 64.5% of subjects in the crossover cohort reported ≥50% pain relief from baseline, with a mean NRS pain score of 3.0 ± 2.4 at 12 months (baseline 7.0 ± 1.0). After receiving CRFA, subjects in the crossover cohort had a 27.5% improvement in total WOMAC score. All subjects receiving CRFA reported significant improvement in quality of life. There were no serious adverse events related to either procedure and overall adverse event profiles were similar. CONCLUSION: A majority of subjects treated with CRFA demonstrated sustained knee pain relief for at least 12-months. Additionally, CRFA provided significant pain relief for HA subjects who crossed over 6 months after treatment. TRIAL REGISTRATION: This trial was registered on ClinicalTrials.gov, NCT03381248. Registered 27 December 2017.

The Risk of Falls After Total Knee Arthroplasty with the Use of a Femoral Nerve Block Versus an Adductor Canal Block: A Double-Blinded Randomized Controlled Study.

BACKGROUND: Adductor canal block (ACB) has emerged as an appealing alternative to femoral nerve block (FNB) that produces a predominantly sensory nerve block by anesthetizing the saphenous nerve. Studies have shown greater quadriceps strength preservation with ACB compared with FNB, but no advantage has yet been shown in terms of fall risk. The Tinetti scale is used by physical therapists to assess gait and balance, and total score can estimate a patient's fall risk. We designed this study to test the primary hypothesis that FNB results in a greater proportion of "high fall risk" patients postoperatively using the Tinetti score compared with ACB. METHODS: After institutional review board approval, informed written consent to participate in the study was obtained. Patients undergoing primary unilateral total knee arthroplasty were eligible for enrollment in this double-blind, randomized trial. Patients received either an ACB or FNB (20 mL of 0.5% ropivacaine) with catheter placement (8 mL/h of 0.2% ropivacaine) in the setting of multimodal analgesia. Continuous infusion was stopped in the morning of postoperative day (POD)1 before starting physical therapy (PT). On POD1, PT assessed the primary outcome using the Tinetti score for gait and balance. Patients were considered to be at high risk of falling if they scored <19. Secondary outcomes included manual muscle testing of the quadriceps muscle strength, Timed Up and Go (TUG) test, and ambulation distance on POD1 and POD2. The quality of postoperative analgesia and the quality of recovery were assessed with American Pain Society Patient Outcome Questionnaire Revised and Quality of Recovery-9 questionnaire, respectively. RESULTS: Sixty-two patients were enrolled in the study (31 ACB and 31 FNB). No difference was found in the proportion of "high fall risk" patients on POD1 (21/31 in the ACB group versus 24/31 in the FNB group [P = 0.7]; relative risk, 1.14 [95% confidence interval, 0.84-1.56]) or POD2 (7/31 in the ACB versus 14/31 in the FNB group [P = 0.06]; relative risk, 2.0 [95% confidence interval, 0.94-4.27]). The average distance of ambulation during PT and time to up and go were similar on POD1 and POD2. Manual muscle testing grades were significantly higher on POD1 in the ACB group when compared with that in the FNB (P = 0.001) (Wilcoxon-Mann-Whitney odds, 2.25 [95% confidence interval, 1.35-4.26]). There were no other differences in postoperative outcomes. CONCLUSIONS: ACB results in greater preservation of quadriceps muscle strength. Although we did not detect a significant reduction in fall risk when compared with FNB, based on the upper limit of the relative risk, it may very well be present. Further study is needed with a larger sample size.

Postbiotic yeast fermentation product supplementation to lactating goats increases the efficiency of milk production by enhancing fiber digestibility and ruminal propionate, and reduces energy losses in methane.

Although in vitro data with mixed ruminal fluid demonstrated positive effects of posbiotic diet (POS) from lactobacilli on measures of fermentation and microbial profiles, there is a paucity of in vivo data with lactating ruminants. The aim of the study was to evaluate the effects of incorporating POS into diets of lactating goats on energy (E) partitioning, carbon (C) and nitrogen (N) balance, and performance. Ten late-lactation Murciano-Granadina goats were used in a crossover design with 26-d periods. Goats in the control diet (CON) were fed daily at the rate of 1 kg alfalfa hay and 1.5 kg concentrate, and the treatment group (POS) was fed CON with the addition of 3.75 g/d of Probisan Ruminants (PENTABIOL S.L., Navarra, Spain). No differences in DMI were detected. However, ruminal fluid propionate and apparent total tract digestibilities of NDF and ADF were greater (18%, 4.7%, and 5.2%, respectively; P < 0.05) in POS compared with the CON diet. Daily partitioning of E to milk and efficiency of ME intake for milk production greater (11% and 3.0%, respectively; P < 0.05) in POS compared with CON. The nonprotein RQ was greater in POS compared with CON due to greater (P < 0.05) oxidation of carbohydrate (213 vs. 115 kJ/kg of BW0.75 per day) compared with fat (362 vs. 486 kJ/kg of BW0.75 per day). Although no differences were found in C balance, goats in POS had lower (P < 0.05) amounts of C in CH4 (1.1 vs. 1.3 g/kg BW0.75 per day) compared with CON. There were no differences in N intake or N in feces or urine, but N in milk was greater (P < 0.05) in POS compared with the CON diet (0.8 vs. 0.7 g/kg BW0.75 per day). Yield of fat-corrected milk (FCM) (3.20 vs. 2.72 kg/d; P < 0.05) and concentration of true protein (3.4 vs. 3.3 kg/d; P < 0.05) and lactose (4.7 vs. 4.5 kg/d; P < 0.05) were greater in POS compared with CON. These responses were accompanied by lower (P < 0.05) urea (12.3 vs. 16.6 mM/L) and ammonia-N (6.6 vs. 8.8 mg/L) without changes in fat concentration (6.1% vs. 6.0%; P > 0.05) in POS compared with the CON diet. Daily amount of CH4 emission did not differ P > 0.05 between diets. However, when expressed relative to unit of edible product, feeding POS reduced (P < 0.05) the amount of CH4 by 46 g/kg of milk fat, 97 g/kg of milk protein, and 3 g/kg of milk compared with CON. Overall, data indicated that feeding a postbiotic in late-lactation increased energy efficiency for milk production partly by reducing CH4 emission.

Although in vitro data with mixed ruminal fluid demonstrated positive effects of postbiotics from lactobacilli on measures of fermentation and microbial profiles, there is a paucity of in vivo data with lactating ruminants. We evaluated the effects of incorporating a postbiotic yeast fermentation product in diets of lactating goats on energy partitioning, carbon and nitrogen balance, and performance. The postbiotic led to greater ruminal propionate concentration and fiber digestibility, and decreased partitioning of energy to methane. Those changes were associated with greater milk production. Data suggested that postbiotics could enhance efficiency of nutrient use for milk production.

Cannabis, cannabinoids, and cannabis-based medicines: future research directions for analgesia.

The use of cannabis spans thousands of years and encompasses almost all dimensions of the human experience, including consumption for recreational, religious, social, and medicinal purposes. Its use in the management of pain has been anecdotally described for millennia. However, an evidence base has only developed over the last 100 years, with an explosion in research occurring in the last 20-30 years, as more states in the USA as well as countries worldwide have legalized and encouraged its use in pain management. Pain remains one of the most common reasons for individuals deciding to use cannabis medicinally. However, cannabis remains illegal at the federal level in the USA and in most countries of the world, making it difficult to advance quality research on its efficacy for pain treatment. Nonetheless, new products derived both from the cannabis plant and the chemistry laboratory are being developed for use as analgesics. This review examines the current landscape of cannabinoids research and future research directions in the management of pain.

Opioid Taper is Associated with Improved Experimental Pain Tolerance in Patients with Chronic Pain: An Observational Study.

INTRODUCTION: The degree to which opioid-induced hyperalgesia contributes to the pain experience of patients with chronic pain remains relatively undescribed. The objective of this pilot study was to determine if experimental pain responses improve in patients with chronic pain as they undergo a planned opioid taper. METHODS: This was a prospective observational study. Seven patients with chronic neuropathic pain on at least 120 mg morphine equivalents/day were enrolled. The participants were followed over the course of an individualized opioid taper to a lower dose. Measures of experimental pain sensitivity, including indicators of central pain modulation, were collected on a biweekly basis; in addition, measures of function and quality of life were collected monthly. The effect of opioid taper on pain responses and functional outcomes over time were examined using longitudinal mixed-effects regression modeling and general linear regression modeling with regularization as a function of baseline dose, end dose, and taper rate. RESULTS: In this small sample of patients undergoing highly individualized and variable opioid taper, the opioid taper was significantly associated with improved pain responses to the cold-pressor test, with the pain threshold on average increasing by 1.14 s every 6 weeks (p = 0.0084, 95% confidence interval [CI] for 6-week change 0.3039-2.0178) and pain tolerance on average increasing by 2.87 s every 6 weeks (p = 0.0026, 95% CI for 6-week change 1.02-4.7277). Taper-related changes in central pain modulation were not observed, although conditioned modulation trended toward improvement by the completion of opioid taper. Similarly, no declines in function and quality of life were observed with the opioid taper, suggesting stability despite decreased opioid dose. CONCLUSIONS: Opioid taper was associated with improvements in experimental pain responses without a decline in function and quality of life, suggestive of diminished opioid-induced hyperalgesia in this clinical sample. TRIAL REGISTRATION: ClinicalTrials.gov identifier, NCT03912298.

Post-dural Puncture Headache After Removal of Trail Spinal Cord Stimulator Leads: A Case Report.

Spinal cord stimulators (SCS) have been an invaluable resource in treating chronic pain pathologies such as failed back surgery syndrome, complex regional pain syndrome, neuropathic pain, and leg ischemia. Post-dural puncture headaches (PDPH) are a common phenomenon that happens when the dura is compromised. It has been seen with permanent SCS placement, but less commonly reported with SCS trail leads. We present a case of a patient who developed PDPH symptoms, not after initial trial leads placement but upon their removal. This case not only illustrates that dural compromise can occur when the placement of the leads is correct with confirmatory imaging, but also the leads themselves can contribute to masking the defect.

Influence of dietary n-3 long-chain fatty acids on microbial diversity and composition of sows' feces, colostrum, milk, and suckling piglets' feces.

Introduction: Very little is known about the impact of n-3 long-chain fatty acids (n-3 LCFAs) on the microbiota of sows and their piglets. The aim of this study was to evaluate the effect of n-3 LCFA in sow diets on the microbiota composition of sows' feces, colostrum, and milk as well as that of piglets' feces. Methods: Twenty-two sows were randomly assigned to either a control or an n-3 LCFA diet from service to weaning. Sows' and piglets' performance was monitored. The gestating and lactating sows' microbiomes in feces, colostrum, and milk were characterized by 16s ribosomal RNA gene sequencing. The fecal microbiome from the two lowest (>800 g) and the two highest birth weight piglets per litter was also characterized, and the LPS levels in plasma were analyzed at weaning. Results and Discussion: n-3 LCFA increased microbiota alpha diversity in suckling piglets' and gestating sows' feces. However, no effects were observed in colostrum, milk, or lactating sows' feces. Dietary n-3 LCFA modified the microbiota composition of gestating sows' feces, milk, and suckling piglets' feces, without affecting lactating sows' feces or colostrum. In gestating sows' feces and milk, the decrease in genus Succinivibrio and the increase of Proteobacteria phylum, due to the increased genera Brenneria and Escherichia, respectively, stand out. In the feces of suckling piglets, the higher abundance of the beneficial genus Akkermansia and Bacteroides, and different species of Lactobacillus are highlighted. In addition, positive correlations for families and genera were found between lactating sows' feces and milk, milk and suckling piglets' feces, and lactating sows' feces and suckling piglets' feces. To conclude, dietary n-3 LCFA had a positive impact on the microbiome of suckling piglet's feces by increasing microbial diversity and some beneficial bacteria populations, had a few minor modifications on the microbiome of milk and gestating sows' feces and did not change the microbiome in lactating sows' feces or colostrum. Therefore, this study shows the effect of dietary n-3 LCFA on the microbiota of sows, colostrum, milk, and suckling piglets during the lactation period providing crucial information on the microbiota status at the early stages of life, which have an impact on the post-weaning.

Color Doppler ultrasonography of an agitated solution is predictive of accurate catheter placement for a continuous popliteal sciatic nerve block.

BACKGROUND: Continuous peripheral nerve catheters (PNCs) have been shown to provide superior postoperative analgesia, decrease opioid consumption, and improve patient satisfaction compared with single injection techniques. In order to achieve success and reliability, accurate catheter positioning is an essential element of PNC placement. An agitated solution of normal saline, D5W, or a local anesthetic solution can be produced by the introduction of air to the injectate, creating air bubbles that can enhance ultrasonographic visualization and possibly improve block success. METHODS: Eighty-three patients were enrolled. Ultrasound-guided continuous popliteal sciatic nerve blocks were performed by positioning the tip of a Tuohy needle between the tibial and common peroneal branches of the sciatic nerve and threading a catheter. An agitated local anesthetic solution was injected through the catheter, viewed with color Doppler ultrasound and video recorded. A peripheral block score (lower score = greater blockade, range 0-14) was calculated based upon the motor and sensory testing at 10, 20, and 30 min after block completion. The color Doppler agitation coverage pattern for the branches of the sciatic nerve was graded as follows: complete (> 50%), partial (> 0%, ≤ 50%), or none (0%). RESULTS: The degree of nerve blockade at 30 min as judged by median (10th, 90th percentile) peripheral block score was significant for partial or complete color Doppler coverage of the sciatic nerve injectate compared to no coverage [3 (0, 7) vs 8 (4, 14); p < 0.01] and block onset was faster (p = 0.03). The block success was higher in groups with partial or complete coverage of the branches of the sciatic nerve vs no coverage (96% vs 70%; p = 0.02). CONCLUSIONS: Injection of an agitated solution through a popliteal sciatic perineural catheter is predictive of accurate catheter placement when partial or complete coverage of the sciatic nerve branches is visualized with color Doppler ultrasound. TRIAL REGISTRATION: NCT01591603.

Analgesic efficacy of adding the IPACK block to a multimodal analgesia protocol for primary total knee arthroplasty.

BACKGROUND AND OBJECTIVES: Peripheral nerve blocks have been integrated into most multimodal analgesia protocols for total knee arthroplasty (TKA). The adductor canal block (ACB) has gained popularity because of its quadriceps muscle sparing. Similarly, local anesthetic injection between the popliteal artery and the posterior capsule of the knee, IPACK block, has been described to provide analgesia to the posterior capsule of the knee with motor-sparing qualities. This prospective randomized controlled trial aimed to assess the analgesic efficacy of adding the IPACK block to our current multimodal analgesic regimen, including the ACB, in patients undergoing primary TKA. METHODS: 119 patients were randomized to receive either an IPACK or a sham block in addition to multimodal analgesia and an ACB. We were set to assess pain in the back of the knee 6 hours after surgery. Other end points included quality of recovery after surgery, pain scores, opioid requirements, and functional measures. RESULTS: Patients who received the IPACK block had less pain in the back of the knee 6 hours after surgery when compared with the sham block: 21.7% vs 45.8%, p<0.01. There was marginal improvement in other pain measures in the first 24 hours after surgery. However, opioid requirements, quality of recovery and functional measures were similar between the two groups. CONCLUSION: The IPACK block reduced the incidence of posterior knee pain 6 hours postoperatively.

Cooled Radiofrequency Ablation Compared with a Single Injection of Hyaluronic Acid for Chronic Knee Pain: A Multicenter, Randomized Clinical Trial Demonstrating Greater Efficacy and Equivalent Safety for Cooled Radiofrequency Ablation.

BACKGROUND: Knee osteoarthritis is a painful and sometimes debilitating disease that often affects patients for years. Current treatments include short-lasting and often repetitive nonsurgical options, followed by surgical intervention for appropriate candidates. Cooled radiofrequency ablation (CRFA) is a minimally invasive procedure for the treatment of pain related to knee osteoarthritis. This trial compared the efficacy and safety of CRFA with those of a single hyaluronic acid (HA) injection. METHODS: Two hundred and sixty subjects with knee osteoarthritis pain that was inadequately responsive to prior nonoperative modalities were screened for enrollment in this multicenter, randomized trial. One hundred and eighty-two subjects who met the inclusion criteria underwent diagnostic block injections and those with a minimum of 50% pain relief were randomized to receive either CRFA on 4 genicular nerves or a single HA injection. One hundred and seventy-five subjects were treated (88 with CRFA and 87 with HA). Evaluations for pain (Numeric Rating Scale [NRS]), function (Western Ontario and McMaster Universities Osteoarthritis Index [WOMAC]), quality of life (Global Perceived Effect [GPE] score and EuroQol-5 Dimensions-5 Level [EQ-5D-5L] questionnaire), and safety were performed at 1, 3, and 6 months after treatment. RESULTS: Demographic characteristics did not differ significantly between the 2 study groups. A total of 158 subjects (76 in the CRFA group and 82 in the HA group) completed the 6-month post-treatment follow-up. In the CRFA group, 71% of the subjects had ≥50% reduction in the NRS pain score (primary end point) compared with 38% in the HA group (p < 0.0001). At 6 months, the mean NRS score reduction was 4.1 ± 2.2 for the CRFA group compared with 2.5 ± 2.5 for the HA group (p < 0.0001). The mean WOMAC score improvement at 6 months from baseline was 48.2% in the CRFA group and 22.6% in the HA group (p < 0.0001). At 6 months, 72% of the subjects in the CRFA group reported improvement in the GPE score compared with 40% in the HA group (p < 0.0001). CONCLUSIONS: CRFA-treated subjects demonstrated a significant improvement in pain relief and overall function compared with subjects treated with a single injection of HA. No serious adverse events related to either procedure were noted, and the overall adverse-event profiles were similar. LEVEL OF EVIDENCE: Therapeutic Level I. See Instructions for Authors for a complete description of levels of evidence.

Top Ten Tips Palliative Care Clinicians Should Know About Interventional Pain and Procedures.

Pain is a common symptom for patients with advanced illness. Palliative care (PC) clinicians are experts in pharmacologic and nonpharmacologic treatment of pain and other symptoms for these patients. True multimodal pain control should include consideration of interventional procedures and pumps to improve difficult-to-manage pain. This article, written by clinicians with expertise in interventional pain and PC, outlines and explains many of the adjunctive and interventional therapies that can be considered for patients with pain in the setting of serious illness. Only by understanding and considering all available options can we ensure that our patients are receiving optimal care.

Music versus midazolam during preoperative nerve block placements: a prospective randomized controlled study.

BACKGROUND AND OBJECTIVES: Music medicine is a non-pharmacologic intervention that is virtually harm-free, relatively inexpensive and has been shown to significantly decrease preoperative anxiety. In this study we aim to compare the use of music to midazolam as a preoperative anxiolytic prior to the administration of an ultrasound-guided single-injection peripheral nerve block. METHODS: In this randomized controlled study we compared the anxiolytic effects of intravenous midazolam (1-2 mg) with noise-canceling headphone-delivered music medicine. All patients received a preoperative ultrasound-guided single-injection peripheral nerve block indicated for a primary regional anesthetic or postoperative analgesia. RESULTS: The change in the State Trait Anxiety Inventory-6 (STAI-6) anxiety scores from after to before the procedure were similar in both groups (music group -1.6 (SD 10.7); midazolam group -4.2 (SD 11); p=0.14; mean difference between groups -2.5 (95% CI -5.9 to 0.9), p=0.1). Patient satisfaction scores with their procedure experience were higher in the midazolam group (p=0.01); however, there were no differences in physician satisfaction scores of their procedure experience between groups (p=0.07). Both patient and physician perceptions on difficulties in communication were higher in the music group than in the midazolam group (p=0.005 and p=0.0007, respectively). CONCLUSIONS: Music medicine may be offered as an alternative to midazolam administration prior to peripheral regional anesthesia. However, further studies are warranted to evaluate whether or not the type of music, as well as how it is delivered, offers advantages over midazolam that outweigh the increase in communication barriers. CLINICAL TRIAL REGISTRY: Clinicaltrials.gov #NCT03069677.

Myxoma virus jumps species to the Iberian hare.

The study of myxoma virus (MYXV) infections in the European rabbit (Oryctolagus cuniculus) has produced one of the most accepted host-pathogen evolutionary models. To date, myxomatosis has been limited to the European rabbit with sporadic reports in hares. However, reports of widespread mortalities in the Iberian hare (Lepus granatensis) with myxomatosis-like clinical signs indicate a potential species jump has occurred. The presence of MYXV DNA was confirmed by PCR in 244 samples received from regional veterinary services, animal health laboratories, hunters or rangers over a 5-month period. PCR analysis of 4 MYXV positive hare samples revealed a 2.8 kb insertion located within the M009 gene with respect to MYXV. The presence of this insertion was subsequently confirmed in 20 samples from 18 Spanish provinces. Sanger sequencing and subsequent analysis show that the insert contained 4 ORFs which are phylogenetically related to MYXV genes M060, M061, M064 and M065. The complete MYXV genome from hare tissue was sequenced using Ion torrent next-generation technology and a summary of the data presented here. With the exception of the inserted region, the virus genome had no large scale modifications and 110 mutations with respect to the MYXV reference strain Lausanne were observed. The next phase in the evolution of MYXV has taken place as a host species jump from the European rabbit to the Iberian hare an occurrence which could have important effects on this naïve population.

A comparison of the fascia iliaca block to the lumbar plexus block in providing analgesia following arthroscopic hip surgery: A randomized controlled clinical trial.

STUDY OBJECTIVE: This randomized controlled single blinded clinical trial compared the fascia iliaca block (FIB) and the lumbar plexus block (LPB) in patients with moderate to severe pain following hip arthroscopic surgery. DESIGN: Single blinded randomized trial. SETTING: Postoperative recovery area, postoperative days 0 and 1. PATIENTS: Fifty patients undergoing hip arthroscopy were approached in the Post Anesthesia Care Unit (PACU) if they had moderate to severe pain (defined as > or equal 4/10 on the numeric rating scale). Twenty-five patients were allocated to the FIB and twenty-five patients to the LPB. INTERVENTIONS: Fascia iliaca block or lumbar plexus block. MEASUREMENTS: A blinded observer recorded pain scores just prior to the block, 15 min following the block (primary endpoint), and then every 15 min for 2 h (or until the patient was discharged). Total PACU time and opioid use were recorded. Pain scores and analgesic use on postoperative day (POD) 0, and POD 1 were recorded. At 24 h post block the Quality of Recovery 9 questionnaire was administered. RESULTS: The mean pre-block pain scores were comparable between the two groups (P = 0.689). There was no difference in mean post block pain scores between the two groups at 15 min (P = 0.054). In the PACU patients who underwent a LPB consumed less opioids compared to FIB patients (P = 0.02), however no differences were noted between the two groups in PACU length of stay, or POD 0 or 1 opioid use. CONCLUSION: A fascia iliaca block is not inferior to a lumbar plexus block in reducing PACU pain scores in patients with moderate to severe pain following hip arthroscopic surgery and is a viable option to help manage postoperative pain following hip arthroscopic surgery.

Non-viability of Haemophilus parasuis fur-defective mutants.

By complementation of an Escherichia coli fur mutant, the Haemophilus parasuis fur gene has been isolated from a genomic library of this organism. The H. parasuis fur gene is the distal one of a three-gene operon. Two genes placed upstream of the H. parasuis fur open-reading frame encode for a hypothetical protein and a flavodoxin, respectively. Attempts performed to isolate an H. parasuis fur-defective mutant either through manganese-resistance selection or exchange markers were unsuccessful. Likewise, anaerobic growth conditions do not enable the attainment of H. parasuis fur-defective mutants either. Nevertheless, H. parasuis clones carrying a knockout mutation in the chromosomal fur gene by insertion of a KmR cassette were obtained when a stable plasmid, containing an additional copy of the transcriptional unit to which the fur gene belongs, was present. Likewise, the presence of a plasmid in which the H. parasuis fur gene is under the control of the Escherichia coli tac promoter allows for the isolation of fur::Km mutants of this organism. Nonetheless, no fur-defective mutants may be isolated from H. parasuis cells harbouring a stable plasmid in which only the single fur gene is contained. These data clearly indicate that H. parasuis cell viability requires the presence of a wild-type fur gene.

Quantification of total bacteria, enterobacteria and lactobacilli populations in pig digesta by real-time PCR.

Jejunum digesta samples were taken from weaning pigs in order to evaluate real-time PCR (qPCR) as a method for quantifying pig gut bacteria. Total bacteria, lactobacilli and enterobacteria were quantified by qPCR and the results were compared with those obtained with traditional methods: 4',6-diamidino-2-phenylindole (DAPI staining) for total bacteria, selective culture for lactobacilli and enterobacteria. Real-time PCR showed higher values in terms of 16S rRNA gene copies than DAPI counts or CFU. Despite the differences, the lactobacilli:enterobacteria ratio was similar between methods (2.5 +/- 0.58 for qPCR and 3.1 +/- 0.71 for selective culture, P = 0.39). Possible reasons for the higher PCR counts are discussed considering both an overestimation with PCR by quantification of dead bacteria or free DNA and also an underestimation with conventional methods. Inherent differences in the pre-treatment of the samples could partially explain the discrepancies observed. Regardless of the numerical differences between methods, values obtained by qPCR and traditional methods showed a significant correlation for lactobacilli and total bacteria. In the light of these results, real-time PCR seems a valid method to quantify microbial shifts in the gastrointestinal tract.

Can Chronic Pain Be Prevented?

All chronic pain begins at some discrete point in time. Significant strides in the understanding of mechanisms and risk factors associated with the transition from a new, or acute, pain experience to a chronic pain condition have been made over the past 20 years. These insights provide the hope of one day being able to modify or even halt this pathophysiologic progression. This article reviews some of the current knowledge of this transition as well as the evidence currently available to best prevent and treat it using persistent surgical pain as a model.

Development of a genetic manipulation system for Haemophilus parasuis.

Haemophilus parasuis is a member of the family Pasteurellaceae and an important respiratory-tract pathogen of swine, which is the etiological agent of Glasser's disease. Because no genetic manipulation system is available for H. parasuis so far, in vivo studies about the role of its genes involved in virulence are unfeasible. Here we demonstrate that H. parasuis has a cyclic AMP (cAMP)-dependent natural transformation system that enables the uptake of DNA in which the ACCGAACTC sequence signal must be present. After improving DNA transformation parameters, such as cAMP and DNA concentration and exposition time of the exogenous DNA, a knockout mutant of H. parasuis defective in the thy gene, encoding the thymidylate synthase enzyme, has been constructed. Data presented in this work open the possibility for the functional analysis of genes involved in the infectious process of this animal pathogen.