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Biomedical subjects

I R Poxton

Publications and source records attributed to I R Poxton.

At least 19 recordsLinked to original sources
PubMed2006

Comparison of IgG antibody levels to Clostridium botulinum antigens between euthanased and surviving cases of chronic grass sickness.

Serum from 12 horses suffering from chronic grass sickness (CGS) were assayed for IgG antibodies against botulinum neurotoxins C and D (BoNT/C and BoNT/D) and to a surface antigen extract of a neurotoxin negative strain of Clostridium botulinum type C. Collectively, the six surviving CGS cases demonstrated significantly higher initial IgG levels (P=0.05) against surface antigens than the six that were subsequently euthanased. The surviving animals also demonstrated higher initial IgG levels against the BoNT/C but not reaching significance (P=0.06). The two groups demonstrated no difference between IgG levels against BoNT/D. This study supports existing evidence of the involvement of C. botulinum type C in the aetiology of grass sickness.

Animals↗
PubMed2004

Association between Key-Gaskell syndrome and infection by Clostridium botulinum type C/D.

There is growing evidence that equine dysautonomia is a toxicoinfection with Clostridium botulinum type C. The possibility that feline dysautonomia has the same aetiology was investigated by attempting to detect botulinum type C neurotoxin in the food, faeces and the contents of the ileum of affected cats, and by serology. The toxin was detected directly in four of eight affected cats and after enrichment in seven of them, and in their dried food. No toxin was detected in healthy control cats or in their tinned food. Recent exposure to the organism was assessed by the detection of immunoglobulin A (IgA) in the faeces of healthy control cats and affected cats. The levels of IgA antibodies to the toxin and to surface antigens of C. botulinum type C in the faeces of the affected cats 14 weeks after the outbreak were significantly higher than in the faeces of the control cats.

Animal Feed↗
PubMed2004

Equine grass sickness is associated with low antibody levels to Clostridium botulinum: a matched case-control study.

REASONS FOR PERFORMING STUDY: Equine grass sickness is a high mortality disease which, despite many years of investigation, is of unknown aetiology. Recent findings indicating that the disease is associated with Clostridium botulinum require support from an epidemiological study that recognises and controls for potential confounders, e.g. age, time of year and premises. HYPOTHESIS: EGS is associated with low antibody levels to C. botulinum antigens. METHODS: A matched case-control study was conducted. Data were collected from 66 histologically confirmed cases of EGS and 132 premises-matched control horses. The probability of EGS in horses was modelled using conditional logistic regression. RESULTS: EGS was significantly associated (age-adjusted P < 0.005) with low antibody levels to each of 3 clostridial antigens; C. botulinum type C and C. novyi type A surface antigens and a C. botulinum type C toxin complex toxoid. These serological risk factors for EGS remained highly significant when entered into multivariable models. This study also identified new horse-level risk factors for EGS; feeding hay or haylage was associated with a decreased risk of disease, change of feed type or quantity during the 14 days prior to disease was associated with increased risk, and the use of an ivermectin anthelmintic at both the ultimate and penultimate treatments was also associated with a significantly increased risk of EGS. CONCLUSIONS: This study provides strong support for the role of C. botulinum in the aetiology of EGS and identifies managemental risk factors for the disease. POTENTIAL RELEVANCE: Increasing anticlostridial antibody levels by vaccination and appropriate managemental interventions may decrease the risk of EGS occurring.

Animals↗
PubMed2003

A European survey of diagnostic methods and testing protocols for Clostridium difficile.

OBJECTIVE: To conduct a survey of the methods used in clinical microbiology laboratories in Europe to diagnose infection with Clostridium difficile. METHODS: A questionnaire was devised and sent to a co-ordinating member of the Study Group in each of eight European countries. This co-ordinator was in charge of forwarding the questionnaire to hospital laboratories arbitrarily selected. The number of laboratories in each country was determined on the basis of one laboratory for 10,000 beds of hospitalization. This questionnaire covered different aspects pertaining to Clostridium difficile associated to diarrhea (CDAD) diagnosis such as circumstances of request, criteria used for undertaking C. difficile investigations, methods used for the diagnosis, etc. RESULTS: A total of 212 questionnaires were completed and submitted for analysis: 87.7% of laboratories reported routinely performing C. difficile diagnostic tests. Methods used included toxin detection (93%), culture (55%), and glutamate dehydrogenase (GDH) detection (5.9%). Among the laboratories detecting toxins, different enzyme immunoassays (EIA) and cytotoxicity assays were used in 79% and 17.3% of cases, respectively. Among the different strategies reported, 4.8% were considered suboptimal for the diagnosis of C. difficile infections, but marked discrepancies could be observed between countries. The overall incidence (median) of CDAD was estimated at 1.1 for 1,000 patient admissions. CONCLUSION: The results of this study suggest marked discrepancies between laboratories and also between countries regarding the criteria by which C. difficile is investigated for, and the methods and the strategies that are used for the diagnosis of C. difficile. These discrepancies could be explained by the lack of clear guidelines for C. difficile diagnosis in each country, and by the importance that physicians attach to C. difficile. Precise guidelines for C. difficile diagnosis would be the first step to make possible accurate comparison of the incidence and the epidemiology of CDAD from one hospital to another or from one country to another.

Bacterial Proteins↗
PubMed2002

Detection of toxin production in Clostridium difficile strains by three different methods.

OBJECTIVE: To compare two immunoassays for detection of toxins produced in vitro by isolates of Clostridium difficile with the standard tissue culture assay, to help in the diagnosis of C. difficile-associated diarrhoea. METHODS: Toxin production was investigated in 42 strains of C. difficile of various serotypes, ribotypes and S-protein types. These included strains from our laboratory collection, strains freshly isolated from stool specimens of patients suspected of suffering from C. difficile-associated disease or of carrying it asymptomatically, and one reference strain (NCTC 11223). Toxin was assayed by (i) a rapid slide immunoassay (C. difficile toxin A test, Clearview, Oxoid), (ii) an enzyme-linked microplate immunoassay (C. difficile toxin A/B test, Techlab), and (iii) a tissue culture assay. The rapid slide assay and the enzyme immunoassay were performed according to the manufacturers' recommendations. The tissue culture assay was performed using Vero cells. RESULTS: Thirty of the 42 strains (71%) were shown to be positive for toxin A by the slide immunoassay and 34 of the strains (81%) were found to be toxin A/B producers by the enzyme immunoassay. The same 34 strains that were positive in the enzyme immunoassay also produced toxin B (cytotoxin) in the tissue culture assay. The sensitivity, specificity, and positive and negative predictive values for the rapid slide immunoassay method were calculated to be 88.2%, 100.0%, 100.0% and 66.7%, respectively, when compared to tissue culture assay results as the reference method. These values for the enzyme immunoassay method were all 100.0%. In this study eight strains were found to be non-toxin-producing by all methods. It is possible that there were four strains that only produced toxin B (A- B+), and were missed by the rapid A-only assay. CONCLUSIONS: We can recommend the use of the Techlab A + B enzyme immunoassay for the detection of toxin production by C. difficile strains because of its high sensitivity and specificity, its ease of use, and its capability of detecting both A- and B-type toxins.

Animals↗
PubMed2001

Survival of Mannheimia (Pasteurella) haemolytica in tracheobronchial washings of sheep and cattle.

The growth, morphology and long-term survival of a representative isolate of Mannheimia haemolytica serotypes A1 and A2 were monitored in ovine and bovine tracheobronchial washings. Both strains survived for at least 244 days in ovine tracheobronchial washings and 156 days in bovine tracheobronchial washings. The addition of fresh washings at these times prompted an increase for serotype A2 but no change in viability for serotype A1 in ovine tracheobronchial washings and an increase for both serotypes in bovine tracheobronchial washings. When growth and survival was compared using tracheobronchial washings from ruminant and non-ruminant species there was a trend towards longer survival in ruminant fluids.Long-term survival was associated with temporary or permanent change from normal size colonies to 'micro-colonies' on sheep blood agar. Subculture allowed reversion to normal colony morphology. Analysis showed these micro-colonies to consist of chains of elongated bacteria. M. haemolytica serotype A2 was more robust in its ability to withstand nutrient deprivation for long periods of time. These survival mechanisms may have important implications for pathogenesis.

Animals↗
PubMed2001

The pathogenicity of Clostridium difficile.

It is now well established that the major virulence factors of C. difficile are the two toxins A and B. However, the organism possesses an array of other putative virulence factors that may be important for localisation within the colon, and in evasion of the immune system. It has been observed that certain types of C. difficile are more commonly found causing disease than others, and this seems to be independent of toxin production. Is this simply a reflection of their abundance in the hospital environment, or is it due to their virulence determinants? This review covers our current knowledge of the modes of action of toxins A and B at the cellular and molecular level. Many unanswered questions are posed that require answers before we can fully understand the pathogenic mechanisms of the organism and be in a position to manage better the spectrum of diseases it causes.

Bacterial Proteins↗
PubMed2001

Variation in the surface layer proteins of Clostridium difficile.

Surface layers (S-layers) form regular crystalline structures on the outermost surface of many bacteria. Clostridium difficile possesses such an S-layer consisting of two protein subunits. Treatment of whole cells of C. difficile with 5 M guanidine hydrochloride revealed two major proteins of different molecular masses characteristic of the S-layer on SDS-PAGE. In this study 25 isolates were investigated. A high degree of variability in the molecular mass of the two S-layer proteins was evident. Molecular masses ranged from 48 to 56 kDa for the heavier protein and from 37 to 45 kDa for the lighter protein. A further protein component of 70 kDa was detectable in all isolates. No cross-reaction was seen between the two major proteins from isolates that produced different S-layer patterns, and most S-layer proteins from isolates with the same or similar banding patterns did not cross-react. The S-layer proteins, when detected by a combination of Coomassie blue staining and immunoblotting, are a useful marker for phenotyping.

Animals↗
PubMed2001

Systemic antibodies to Clostridium botulinum type C: do they protect horses from grass sickness (dysautonomia)?

The aetiology of equine grass sickness (EGS) is still unknown. There is increasing evidence that toxicoinfection with Clostridium botulinum type C is involved. Epidemiological evidence shows that resistance to EGS can occur in older horses and those that have been on a particular pasture for longer or have been in prior contact with the disease. This resistance may be in the form of an immune response to the aetiological agent. Levels of systemic antibodies to the surface antigens of C. botulinum type C (using the closely related and safe C. novyi type A as a phenotypic marker) and to the botulinum type C neurotoxin (BoNT/C) were investigated in horses with and without EGS. Horses with grass sickness were found to have significantly lower levels of systemic IgG to both surface antigens and BoNT/C. Horses with low levels of systemic immunity to these antigens may be more susceptible to developing EGS. There were no significant differences in antibody levels between the different categories of EGS, suggesting systemic immunity to C. botulinum type C does not play a significant role in influencing the severity of the disease. However, horses that had been in contact with EGS or that were grazing land where it had occurred frequently in the past had significantly higher antibody levels to these antigens. These horses may have been exposed to subclinical doses of C. botulinum type C and BoNT/C, resulting in the production of a protective immune response against the putative aetiological agent. This finding is of potential significance for the prospect of prevention of EGS by vaccination against C. botulinum type C.

Age Factors↗
PubMed2000

Endotoxin and nanobacteria in polycystic kidney disease.

BACKGROUND: Microbes have been suspected as provocateurs of polycystic kidney disease (PKD), but attempts to isolate viable organisms have failed. Bacterial endotoxin is the most often reported microbial product found in PKD fluids. We assessed potential microbial origins of endotoxin in cyst fluids from 13 PKD patients and urines of PKD and control individuals. METHODS: Fluids were probed for endotoxin and nanobacteria, a new bacterium, by the differential Limulus Amebocyte Lysate assay (dLAL), genus-specific antilipopolysaccharide (LPS) antibodies, monoclonal antibodies to nanobacteria, and hyperimmune serum to Bartonella henselae (HS-Bh). Selected specimens were also assessed by transmission electron microscopy (TEM) and nanobacterial culture methods. RESULTS: LPS or its antigenic metabolites were found in more than 75% of cyst fluids tested. Nanobacteria were cultured from 11 of 13 PKD kidneys, visualized in 8 of 8 kidneys by TEM, and immunodetected in all 13 PKD kidneys. By immunodetection, nanobacterial antigens were found in urine from 7 of 7 PKD males, 1 of 7 PKD females, 3 of 10 normal males, and 1 of 10 normal females. "Nanobacterium sanguineum" was dLAL positive and cross-reactive with antichlamydial LPS and HS-Bh. Some cyst fluids were also positive for LPS antigens from Escherichia coli, Bacteroides fragilis and/or Chlamydia, and HS-Bh, as were liver cyst fluids from one patient. Tetracycline and citrate inhibited nanobacterial growth in vitro. CONCLUSION: Nanobacteria or its antigens were present in PKD kidney, liver, and urine. The identification of candidate microbial pathogens is the first step in ascertaining their contribution, if any, to human disease.

Adult↗
PubMed2000

Exposure to bacteroides fragilis endotoxin during cardiac surgery.

UNLABELLED: Although endotoxemia has been observed during cardiac surgery, the identity of endotoxins to which patients are exposed is unknown. We tested the hypothesis that antibodies to Bacteroides fragilis (an anaerobic gut commensal and a common pathogen) decrease during cardiac surgery, thereby reflecting systemic exposure to this type of endotoxin. Serum antiendotoxin antibody levels were measured in 55 patients during routine cardiac surgery at the following times: Preoperatively, Pre-CPB (immediately before initiation of cardiopulmonary bypass [CPB]), Pre-CPB+5 (5 min after initiation of CPB), and End (end of surgery). Antiendotoxin antibody levels were determined by using enzyme-linked immunosorbent assay. Total immunoglobulin M (IgM) levels were measured by using laser nephelometry and decreases in total IgM levels were used to control changes in antiendotoxin antibody levels attributable to hemodilution. Median (interquartile range) hemodilution corrected IgM anti-B fragilis antibody levels decreased by 12% (5%-20%) from Preoperatively to End of surgery (P < 0.001). In contrast, median hemodilution corrected anti-B fragilis antibody levels did not change significantly from Pre-CPB to Pre-CPB+5, validating the correction for hemodilution. Immunoglobulin G anti-B fragilis antibody levels and IgM and immunoglobulin G anticore antibody levels decreased similarly during surgery. Intraoperatively, levels of anti-B fragilis endotoxin antibodies decreased significantly out of proportion to hemodilution. These results suggest that cardiac surgical patients are exposed to B fragilis endotoxin. IMPLICATIONS: We prospectively measured hemodilution-corrected antiendotoxin antibody levels in 55 cardiac surgical patients. We observed significant decreases in hemodilution-corrected levels of antibody to both Bacteroides fragilis and the core of endotoxin.

Adult↗
PubMed2000

Differences in intestinal humoral immunity between healthy volunteers from UK and Bangladesh.

BACKGROUND/AIMS: Intestinal morphology has been shown to vary geographically. The impact of this variation on gut mucosal humoral immunity is not well-studied. The technique of peroral whole-gut lavage (WGL) with nonabsorbable cleansing fluid can be utilized for the study of gut immune responses in health and disease. In this study, the WGL technique was employed to compare various gut humoral immune parameters in healthy volunteers from Dhaka in Bangladesh and Edinburgh, UK. METHODS: Eleven healthy individuals (all male, age range 18-32) from Dhaka and 12 healthy individuals (4 male and 8 female, age range 23-48) from Edinburgh underwent WGL with a polyethylene glycol electrolyte-based solution drunk at a rate of 1 l/h. The first clear effluent was collected and processed. An ELISA technique was used to measure total immunoglobulins (A, M and G) and antibodies to bacterial lipopolysaccharide (LPS: endotoxin) ovalbumin and eotaxin. Immunoturbimetry and radioimmunoassy techniques were used to measure protein (albumin and alpha-1 antitrypsin) and eosinophil cationic protein (ECP), respectively, in WGL fluid (WGLF). RESULTS: The total IgA, ECP and eotaxin concentrations in WGLF from the Dhaka group were significantly higher than those of the Edinburgh group (P < 0.03, P < 0.002 and P< 0.005 respectively). The IgA antibody level against the core oligosaccharide of bacterial LPS from several Gram-negative species was significantly higher in the Dhaka group compared to the Edinburgh group (P< 0.0001). Similarly, there was generally higher level of IgA antibody response against the various different LPS core structures of Escherichia coli in the Dhaka group, in particular significantly higher against R1, R3 and R4 LPS cores (P< 0.02, P< 0.03 and P< 0.01 respectively) compared to the Edinburgh group. In contrast to antibacterial antibodies, the IgA and IgM antibodies against ovalbumin were significantly lower in the Dhaka group (P< 0.001 and P< 0.003, respectively) compared to the Edinburgh group. CONCLUSIONS: This study on gut mucosal humoral immunity from two geographically distinct populations suggests that place of residence influences gut mucosal humoral immunity. This difference in stimulation of humoral immunity of the gut might explain different rates of inflammatory bowel diseases in developing and developed countries, and also provides a major challenge for the development of mucosally presented vaccine worldwide.

Adult↗
PubMed2000

Endotoxic activity of lipopolysaccharides isolated from emergent potential cystic fibrosis pathogens.

Improved antimicrobial therapies against the classical spectrum of pathogenic bacteria which colonise the lungs of cystic fibrosis (CF) patients has resulted in improved life expectancy and quality of life. Bacterial species that are resistant to a broad range of antibiotics including Stenotrophomonas maltophilia and Alcaligenes xylosoxidans have now emerged as potential new pathogens to fill the niche. At present, it is unclear from clinical data whether these microbes are commensal or pathogenic. In this study we have quantified the inflammatory potential of lipopolysaccharide (LPS) from eight species of Gram-negative organisms which have been cultured with increasing frequency from CF patients. Inflammatory responses induced by LPS from whole human blood and a human-derived monocyte cell line (THP-1) were assessed. Enzyme-linked immunosorbent assays were used to detect interleukin-6, interleukin-8, and tumour necrosis factor alpha (TNF). A bioassay was also used to assess TNF activity. With the exception of S. maltophilia, LPS extracted from all of the bacteria tested upregulated, by varying degrees, expression of each of the proinflammatory cytokines assayed. This study represents the first comprehensive report of the endotoxic potential of a new wave of microbes which are associated with CF.

Cell Line↗
PubMed2000

Preparation and preclinical evaluation of a novel liposomal complete-core lipopolysaccharide vaccine.

Our objective is to develop a prophylactic vaccine strategy that can be evaluated for surgical and other high-risk hospitalized patients. In this paper, we describe the preparation and preclinical evaluation of a liposomal complete-core lipopolysaccharide (LPS) vaccine that is nontoxic and broadly antigenic. Complete-core (Ra-chemotype) LPSs were isolated from four gram-negative bacterial strains (Escherichia coli K-12, E. coli R1, Pseudomonas aeruginosa PAC608, and Bacteroides fragilis), mixed together to form a cocktail of complete-core LPSs, and then incorporated into multilamellar liposomes consisting of dimyristoyl phosphatidyl choline, dimyristoyl phosphatidylglycerol, and cholesterol in a 4:1:4 molar ratio. The endotoxic activities of these LPS-containing liposomes were less than 0.1% of the endotoxicities of the original free LPSs as measured by the Limulus amoebocyte lysate assay. In vivo administration of liposomal complete-core LPS mixed with Al(OH)(3) to rabbits resulted in no pyrogenicity or overt toxicity over a 7-day period. In immunoblots, sera from rabbits following active immunization elicited cross-reactive antibodies to a large panel of rough and smooth LPSs from numerous clinically relevant gram-negative bacteria, including E. coli (serotypes O1, O4, O6, O8, O12, O15, O18, O75, O86, O157, and O111), P. aeruginosa (Fisher-Devlin serotypes 1, 2, and 3, which correspond to International Antigenic Typing Scheme types 6, 11, and 2, respectively), Klebsiella pneumoniae (serotypes O1, O2ab, and O3), B. fragilis, and Bacteroides vulgatus. Active immunization of mice with liposomal complete-core LPS provided protection against a lethal challenge with E. coli O18 LPS. The vaccine tested was nontoxic, nonpyrogenic, and immunogenic against a wide variety of pathogens found in clinical settings.

Animals↗
PubMed1999

The lipopolysaccharide core type of Escherichia coli O157:H7 and other non-O157 verotoxin-producing E. coli.

A mouse monoclonal antibody specific for the R3 lipopolysaccharide core type of Escherichia coli was used to determine the core type of E. coli O157:H7 and other non-O157 verotoxin-producing E. coli strains. Lipopolysaccharide extracts from 28 clinical isolates were examined by sodium dodecylsulfate-polyacrylamide gel electrophoresis and immunoblotting and all were found to have the R3 core. None of the core lipopolysaccharide from the strains tested reacted with the control R1 and R2 specific monoclonal antibodies. A common core type between all the verotoxin-producing E. coli strains tested may be significant when considering the immune response to these bacteria, and to the receptor for the VT bacteriophage.

Animals↗
PubMed1999

The association of Clostridium botulinum type C with equine grass sickness: a toxicoinfection?

The cause of grass sickness, an equine dysautonomia, is unknown. The disease usually results in death. Gastrointestinal (GI) dysfunction is a common clinical manifestation in all forms of the disease. It is generally thought that equine grass sickness (EGS) is caused by an ingested or enterically produced neurotoxin which is absorbed through the GI tract. Clostridium botulinum was first implicated as a causative agent when it was isolated from the GI tract of a horse with EGS in 1919. The aim of the present study was to investigate the hypothesis that EGS results from toxicoinfection with C. botulinum type C: growth of the bacterium in the GI tract with production of toxin (BoNT/C). Ileum contents and faeces from horses with EGS were investigated for BoNT/C, and indirectly for the presence of C. botulinum type C, and compared with control samples from horses without EGS. BoNT/C was detected directly by ELISA in the ileum of 45% (13/29) of horses with EGS compared to 4% (1/28) of controls, and in the faeces of 44% (20/45) of horses with EGS compared to 4% (3/77) of controls. Levels of up to 10 Mlg toxin/g wet weight of gut contents were observed. The one control horse with detectable toxin in the ileum had been clinically diagnosed as having acute EGS, but this was not confirmed by histopathology. The organism was detected indirectly by assaying for BoNT/C by ELISA after enrichment in culture medium. C. botulinum type C was shown to be present in 48% (14/29) of ileum samples and 44% (20/45) of faecal samples from horses with EGS, compared with 7% (2/27) of ileum samples and 8% (6/72) of faecal samples from controls. These results support the hypothesis that EGS results from a C. botulinum type C toxicoinfection.

Animals↗
PubMed1998

Differential effects of bacterial lipopolysaccharides upon neutrophil function.

Lipopolysaccharide (LPS) is a potent inflammatory agent which augments neutrophil sensitivity to subsequent inflammatory stimuli. In this study, the effects of structurally different LPS types upon neutrophil effector functions were examined. Rough LPS types, which have lost the O-polysaccharide moiety, were found to act more rapidly than smooth LPS types in stimulating neutrophil beta2 integrin activity and fMLP-induced respiratory burst. These findings suggest an involvement of the O-polysaccharide region of LPS in regulating neutrophil responsiveness to different LPS chemotypes with important implications for the mechanisms underlying regulation of the inflammatory response in conditions associated with elevation of LPS in plasma, e.g. septic shock or acute respiratory distress syndrome.

CD18 Antigens↗