Evaluation of novel multiplex qPCR assays for diagnosis of pathogens associated with the bovine respiratory disease complex.

Bovine respiratory disease complex is the most common disease requiring the use of antimicrobials in industrial calf production worldwide. Pathogenic bacteria (Mannheimia haemolytica (Mh), Pasteurella multocida (Pm), Histophilus somni (Hs), and Mycoplasma bovis) and a range of viruses (bovine respiratory syncytial virus, bovine coronavirus, bovine parainfluenza virus type 3, bovine viral diarrhea virus and bovine herpesvirus type 1) are associated with this complex. As most of these pathogens can be present in healthy and diseased calves, simple detection of their presence in diseased calves carries low predictive value. In other multi-agent diseases of livestock, quantification of pathogens has added substantially to the predictive value of microbiological diagnosis. The aim of this study was to evaluate the ability of two recently developed quantitative PCR (qPCR) kits (Pneumo4B and Pneumo4V) to detect and quantify these bacterial and viral pathogens, respectively. Test efficiencies of the qPCR assays, based on nucleic acid dilution series of target bacteria and viruses, were 93-106% and 91-104%, respectively, with assay detection limits of 10-50 copies of nucleic acids. All 44 strains of target bacteria were correctly identified, with no false positive reactions in 135strains of non-target bacterial species. Based on standard curves of log10 CFU versus cycle threshold (Ct) values, quantification was possible over a 5-log range of bacteria. In 92 tracheal aspirate samples, the kappa values for agreement between Pneumo4B and bacterial culture were 0.64-0.84 for Mh, Pm and Hs. In an additional 84 tracheal aspirates, agreement between Pneumo4B or Pneumo 4V and certified diagnostic qPCR assays was moderate (0.57) for M. bovis and high (0.71-0.90) for viral pathogens. Thus Pneumo4 kits specifically detected and quantified the relevant pathogens.

Genotyping of single spore isolates of a Pasteuria penetrans population occurring in Florida using SNP-based markers.

AIMS: To generate single spore lines of a population of bacterial parasite of root-knot nematode (RKN), Pasteuria penetrans, isolated from Florida and examine genotypic variation and virulence characteristics exist within the population. METHODS AND RESULTS: Six single spore lines (SSP), 16SSP, 17SSP, 18SSP, 25SSP, 26SSP and 30SSP were generated. Genetic variability was evaluated by comparing single-nucleotide polymorphisms (SNPs) in six protein-coding genes and the 16S rRNA gene. An average of one SNP was observed for every 69 bp in the 16S rRNA, whereas no SNPs were observed in the protein-coding sequences. Hierarchical cluster analysis of 16S rRNA sequences placed the clones into three distinct clades. Bio-efficacy analysis revealed significant heterogeneity in the level virulence and host specificity between the individual clones. CONCLUSIONS: The SNP markers developed to the 5' hypervariable region of the 16S rRNA gene may be useful in biotype differentiation within a population of P. penetrans. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates an efficient method for generating single spore lines of P. penetrans and gives a deep insight into genetic heterogeneity and varying level of virulence exists within a population parasitizing a specific Meloidogyne sp. host. The results also suggest that the application of generalist spore lines in nematode management may achieve broad RKN control.

House dust mite control measures for asthma.

BACKGROUND: The major allergen in house dust comes from mites. Chemical, physical and combined methods of reducing mite allergen levels are intended to reduce asthma symptoms in people who are sensitive to house dust mites. OBJECTIVES: To assess the effects of reducing exposure to house dust mite antigens in the homes of people with mite-sensitive asthma. SEARCH STRATEGY: Cochrane Airways Group trials register, and PubMed and The Cochrane Library (last searches June 2004), reference lists. SELECTION CRITERIA: Randomised trials of mite control measures vs placebo or no treatment in asthmatic people known to be sensitive to house dust mites. DATA COLLECTION AND ANALYSIS: Two reviewers applied the trial inclusion criteria, assessed their quality and extracted the data independently. Study authors were contacted to clarify information. MAIN RESULTS: Forty-nine trials (2733 patients) were included; the number of patients has more than doubled since the last version of this review. Thirty-one trials assessed physical methods, ten assessed chemical methods, and eight a combination of chemical and physical methods. Despite the fact that many trials were of poor quality and would be expected to exaggerate the reported effect, we did not find an effect of the interventions. For the most frequently reported outcome, peak flow in the morning (1339 patients), the standardised mean difference was -0.02 (95% confidence interval (CI) -0.13 to 0.08). There were no statistically significant differences either in number of patients improved (relative risk 1.01, 95% CI 0.80 to 1.27), asthma symptom scores (standardised mean difference -0.01, 95% CI -0.10 to 0.13), or in medication usage (standardised mean difference -0.05, 95% CI -0.18 to 0.09). REVIEWERS' CONCLUSIONS: Chemical and physical methods aimed at reducing exposure to house dust mite allergens cannot be recommended. It is doubtful whether further studies, similar to the ones in our meta-analysis, are worthwhile. If other types of studies are considered, they should be methodologically rigorous and use other methods than those used so far, with careful monitoring of mite exposure and relevant clinical outcomes.

Detection of Pasteuria penetrans infection in Meloidogyne arenaria race 1 in planta by polymerase chain reaction.

We report on the development of a PCR-based assay to detect Pasteuria penetrans infection of Meloidogyne arenaria in planta using specific primers for recently sequenced sigE, spoIIAB and atpF genes of P. penetrans biotype P20. Amplification of these genes in crude DNA extracts of ground tomato root galls using real-time kinetic PCR distinguished infected from uninfected M. arenaria race 1 by analysis of consensus thresholds for single copy genes. Fluorescent in situ hybridization (FISH) using the sigE primer sequence as a probe shows hybridization to P. penetrans cells in various stages of vegetative (pre-endospore) development. Ratios of gene copies for sigE and 16S rDNA were obtained for P. penetrans and compared to Bacillus subtilis as a genomic paradigm of endospore-forming bacteria. Phylogenetic analysis of the sigE gene from Gram-positive, endospore-forming bacteria finds P. penetrans most closely related Paenbacillus polymyxa. The sporulation genes (spo genes), particularly sigE, have sequence diversity that recommends them for species and biotype differentiation of the numerous Pasteuria isolates that infect a large number of plant-parasitic nematodes.

Pasteuria spp.: Systematics and Phylogeny of These Bacterial Parasites of Phytopathogenic Nematodes.

Pasteuria spp. include endospore-forming bacterial pathogens of cladoceran crustaceans and plant-parasitic nematodes. Propagation of these nematode pathogens requires attachment of soilborne endospores to nematode hosts, infection, growth, sporulation, and release of endospores to repeat the cycle of infection and propagation. The ability of these bacteria to suppress the levels of plant-parasitic nematodes in the field has made them particularly promising candidates for biocontrol of nematode diseases of plants. Genes encoding 16S ribosomal RNA have been sequenced for the cladoceran (water flea) parasite and type species, Pasteuria ramosa, and for Pasteuria spp. isolated from root-knot (Meloidogyne arenaria race 1 and Meloidogyne sp.), soybean cyst (Heterodera glycines), and sting (Belonolaimus longicaudatus) nematodes. These have provided a phylogenetic basis for their designation to a distinct clade within the family Alicyclobacillaceae of the gram-positive endospore-forming bacteria. Two apparent biotypes of P. penetrans demonstrating a host preference for different Meloidogyne spp. showed identical 16S rDNA sequences, suggesting host-recognition evolves within a given species. The sequences of genes encoding sporulation transcription factors, sigE and sigF, from P. penetrans biotype P-20 show different phylogenetic relationships to other endospore-forming bacteria, supporting their application to further discriminate Pasteuria spp. and biotypes. Distribution of an adhesin-associated epitope on polypeptides from different Pasteuria isolates provides an immunochemical approach to differentiate species and biotypes with specific host preferences. Application of bioinformatics to genomic data, as well as further characterization of the biochemical basis for host recognition, will facilitate development of Pasteuria spp. as benign alternatives to chemical nematicides.

Environmental quantification of Pasteuria penetrans endospores using in situ antigen extraction and immunodetection with a monoclonal antibody.

Abstract Pasteuria penetrans is an obligate parasite of root-knot nematodes (Meloidogyne spp.) that has attracted significant attention as a promising biocontrol agent. The inability to culture P. penetrans has invoked the need for a quantitative detection capability to facilitate biocontrol studies. A chemical extraction method using urea, dithiothreitol and CHES buffer (UDC) is shown to release soluble endospore envelope antigen from endospores present in complex matrices, generating an extract that can be used to determine the levels of spores when compared to a standard in an enzyme-linked immunosorbent assay (ELISA) using a specific monoclonal antibody, MAb 2A41D10. Extractions can be performed in less than 1 h. Linear regression analysis routinely produced line fits with r(2)>0.90. Antigen extraction efficiency was not influenced by soil type. Three ELISA formats were analyzed for quantitative detection of P. penetrans endospores. A tertiary ELISA immunodetection system provided the lowest level of detection at approximately 300 spores per gram of soil. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis Western blots of soil extracts containing P. penetrans endospore antigen produced signature peptides bearing a common epitope characteristic of endospores of Pasteuria spp. MAb 2A41D10 was specific for Pasteuria spp. and did not react with extracts of Pasteuria-free soil or with spore extracts of native Gram-positive endospore-forming bacteria. Immunofluorescent microscopy revealed that MAb 2A41D10 recognizes an epitope uniformly distributed on the endospore surface. The development of a rapid extraction method and analysis of solubilized antigen by immunodetection has the potential for broad application in food and environmental microbiology.

Biodegradation of low aqueous concentration pentachlorophenol (PCP) contaminated groundwater.

Bioremedial treatment to remove low level organic contamination to regulatory standards has met with limited success. In this study source water from a contaminated surficial aquifer at a former wood treatment facility was used to evaluate the potential for indigenous microorganisms to degrade low level (< 1.0 mg) pentachlorophenol (PCP) to a regulatory drinking water standard of 0.001 mg/L. PCP degradation was evaluated in series of batch reactors in a two phase study to (a) determine the rate and extent of PCP removal and (b) evaluate the impact of nutrient amendment (N and P) on removal rate. All reactors with the exception of the abiotic control demonstrated PCP removal to a level < 0.002 mg/L within a maximum period of 32 d with and without nutrient amendment. A regression analysis of reactive phosphate (ortho-P) concentration versus removal rate produced an R2 of 0.94 (p = 0.006) indicating a significant correlation between the level of available phosphate and PCP degradation rate. Selective bacterial enumeration (for PCP degrading bacteria) revealed PCP-degrading bacteria increased in abundance prior to and in conjunction with the degradation phase to a density of between 10(3) to 10(4) CFU/ml. Isolates were also analyzed for total fatty acids using Fatty Acid Methyl Ester (FAME) methodology and the results indicated that PCP degrading bacteria were present in the aquifer and consisted of predominately fluorescent, oxidase positive Pseudomonas species. Overall, data indicate that autochthonous microbes are capable of removing low level PCP (< 1.0 mg/L) to approach if not reach the regulatory standard of 0.001 mg/L with the addition of oxygen, with or without nutrient amendment. Results of this research can be applied to full-scale implementation of in-situ or ex-situ bioremediation of groundwater at former wood treatment facilities.

Roentgenographic densitometry of bone adjacent to a femoral prosthesis.

A digital imaging method was developed to quantitate the stress-related changes in roentgenographic bone density after total hip arthroplasty. A technique termed "histogram-directed equalization" was used to compensate for differences in postimaging data caused by the effects of variable quality obtained from ten patients. Quantitative change due to variation in delivered energy was decreased by 7% for roentgenograms obtained with a 2 kVp variation and 31% for roentgenograms obtained with a 4 kVp variation. The method allowed the authors to accurately describe the changes observed on annual postoperative roentgenograms obtained over the past decade. The utility of the method was demonstrated in 15 of the senior author's long-term cases treated with fully porous-coated implants. These cases were divided into two groups based on the diameter of the prosthetic stem implanted in each case. Five patients were grouped with small-diameter stems and ten with large-diameter stems. Both groups showed substantial decrease in roentgenographic bone density (from 11% to 28%) in the medial and lateral proximal regions. The large-diameter group had an overall larger decrease in roentgenographic bone density at two and five years. Roentgenographic bone remodeling changes were most pronounced in the first two years. Changes between two and five years progressed at a slower rate. The results also confirmed the predicted effect of stem diameter on bone remodeling patterns.

Home visit effectiveness for peritoneal dialysis patients.

Medicare regulations require follow-up home visits to home dialysis patients, yet routine home visits require a lot of personnel time. The effectiveness of home visits was evaluated by a nurse, dietitian and social worker using a questionnaire. Thirty-six patients were evaluated during an 18-month period. Collectively the team documented an average of 10 pertinent observations per visit and made an average of 4 recommendations for change. Staff members gained new information about the patient as indicated by the fact that they changed their rankings on 5 of 15 parameters following the home visit. The home visit policy that recommended an annual home visit was revised to recommend a single home visit for each new peritoneal dialysis patient. Further visits are performed only if significant problems are identified.

The peritoneal equilibration test: a nursing discussion.

The peritoneal equilibration test is used to assess peritoneal membrane permeability. Results provide a reliable estimate of peritoneal clearances and ultrafiltration rates. This information can be used to make decisions about the preferred dialysis prescription. Repeat tests may be used to evaluate clinical changes. The first part of this two-article series will discuss the clinical interpretation of findings and three case studies. The second article will describe the procedure.

How to do a peritoneal equilibration test.

This article, part 2 of a 2-part series, describes the process and procedures used to perform a peritoneal equilibration test. Data from the test may be used to assess the peritoneal membrane permeability. Knowledge of peritoneal membrane permeability may be useful in selecting a treatment regimen and in determining the appropriate dialysis prescription.

What effect does glucose have on measured creatinine in the peritoneal equilibration test?

Very high glucose concentrations falsely elevate measured creatinine. An appropriate correction factor may be applied to obtain an accurate creatinine value; however, the glucose interference is of a low magnitude in most dialysate samples at a 4-hour dwell time. Clinical interpretation of the peritoneal equilibration test using uncorrected creatinine values to determine the dialysate-to-plasma ratio at 4 hours is associated with minimal error.

Chronic nightly tidal peritoneal dialysis.

Nightly tidal peritoneal dialysis (NTPD) is a technique in which, after an initial fill of the peritoneal cavity, only a portion of dialysate is rapidly cycled. Five anuric, stable, PD patients entered a 4 month study to determine the NTPD session length necessary for clinically adequate dialysis and creatinine clearance similar to those on four daily 2 L CAPD exchanges. NTPD was performed using a modified PAC-X-2 cycler, with the drain phase regulated by a target volume. One patient completed 3.5 months of study, one 4 months, three 6 months, and one patient each continued on NTPD for 13, 14, and 32 months. The mean NTPD session time was 9 hr 24 min (range 8 hr 35 min to 9 hr 55 min) at the end of 4 months. All patients had clinically adequate dialysis. Three patients preferred NTPD over CAPD, particularly because of an empty abdomen during the daytime. One patient required an increase in NTPD time, and an addition of one daytime exchange, because of low creatinine clearance. In conclusion, NTPD provides weekly creatinine clearances comparable to CAPD, with an acceptable duration of nightly dialysis sessions in most anuric patients. A new PD machine providing inexpensive dialysis solution in large quantities, as well as safe and false alarm free dialysis sessions, is needed for practical NTPD implementation.

Tidal peritoneal dialysis with racemic or L-lactate solutions.

UNLABELLED: To see if rapid lactate absorption on tidal peritoneal dialysis (TPD) would overwhelm D-lactate metabolism using racemic lactate and/or L-lactate metabolism using all L-lactate, five patients underwent 8-h TPD treatments with racemic lactate solution one day and with L-lactate another. Lactate concentrations (total) were 40 mmole/L, flow rates 27.3 L/8 h, tidal and reservoir volumes each 1.5L, tidal cycles 24-26 min, and net ultrafiltration per tidal cycle 70 to 99 mL. RESULTS: Mean absorptions of D and L-lactate were 24.2 and 25.1%, respectively, compared to glucose at 14.6%. Urea clearances averaged 21.4 mL/min. Mean blood D-lactates at baseline were 0.6 +/- 0.5 SD mmole/L and after 8 h of TPD were 0.6 +/- 0.4 and 0.7 +/- 0.3 using L-lactate and racemic solutions, respectively; similar values for L-lactate were 1.2 +/- 0.3 at baseline and 1.2 +/- 0.3 and 1.2 +/- 0.5 after 8 h with L-lactate and racemic solutions. delta blood pH values were + 0.02 +/- 0.01 and + 0.04 +/- 0.03, while delta bicarbonate values were + 1.7 +/- 0.9 and + 0.7 +/- 1.0 for the all L and racemic studies, respectively. The total mmoles of L-lactate absorbed per 8 h of TPD with all L solution (greater than 300 mmoles) are greater than ever reported for peritoneal dialysis, but did not increase blood lactate levels. It would seem that either type of solution is suitable for TPD. Absorptions and metabolic rates are similar for L-Lactate and D-Lactate.

Long-term increase in peritoneal membrane transport rates following incidental intraperitoneal sodium hypochlorite infusion.

A patient on continuous ambulatory peritoneal dialysis using an "O" set connection system with sodium hypochlorite as a disinfectant incidentally infused the disinfectant intraperitoneally on two occasions. The product of peritoneal membrane permeability and peritoneal membrane surface area increased after both infusions as judged by peritoneal equilibration test results and/or serum chemistries. Elevated peritoneal solute transport rates and reduced ultrafiltration gradually subsided but did not return to preinfusion values. This observation suggests that intraperitoneal sodium hypochlorite infusion may cause significant long-term alteration in peritoneal membrane transport characteristics.

Effectiveness of a phosphorous educational program for dialysis patients.

Fifty-four percent of all dialysis patients followed by a single center had elevated serum phosphorus levels on more than 25% of all measurements. A phosphorus patient education program was developed and implemented and knowledge was measured with a pretest and posttest. The continuous ambulatory peritoneal dialysis group had a significant increase in knowledge and a minor, but statistically significant, decrease in serum phosphorus after participating in the education program. In contrast, center hemodialysis patients did not demonstrate a significant increase in knowledge or decrease in serum phosphorus. These different outcomes could not be attributed to specific differences between the two groups. Serum phosphorus control is a complex process, and this education program did not result in a clinically significant improvement in serum phosphorus levels.