title: Blastocystis specific serum immunoglobulin in patients with irritable bowel syndrome (IBS) versus healthy controls.
abstract: BACKGROUND
  Blastocystis species are common enteric human parasites and carriage has been linked to Irritable Bowel Syndrome (IBS), particularly diarrhoea-predominant IBS. The spectrum of immune reactivity to Blastocystis proteins has been reported previously in symptomatic patients. We investigated differences in serum immunoglobulin profiles between patients with IBS, both positive and negative for Blastocystis carriage, and healthy controls (HC).
  
  
  METHODS
  Forty diarrhoea-predominant IBS patients (26 patients positive for Blastocystis sp., 14 negative patients) and forty HC (24 positive, 16 Blastocystis-negative) were enrolled. Age, gender, ethnicity and serum immunoglobulin A (IgA) levels were recorded and faecal specimens were analysed using smear, culture and polymerase chain reaction amplification of ribosomal DNA. Sera were tested in Western blots and the reactivities compared to known targets using monoclonal antibodies Blastofluor® (Blastocystis specific antibody), MAb1D5 (cytopathicto Blastocystis cells), anti-promatrix metalloprotease-9 (anti-MMP-9) and SDS-PAGE zymograms.
  
  
  RESULTS
  Levels of serum IgA were significantly lower in Blastocystis carriers (p < 0.001) but had no relationship to symptoms. Western blots demonstrated serum IgG antibodies specific for Blastocystis proteins of 17,27,37,50,60-65, 75-90, 95-105 and 150 kDa MW. Reactivity to the 27, 50 and 75-95 kDa proteins were found more frequently in the IBS group compared to the HC's (p < 0.001) and correlation was greater for Blastocystis-positive IBS patients (p < 0.001) than for negative IBS patients (p < 0.05). MAb1D5 reacted with proteins of 27 and 100 kDa, and anti-MMP-9 with 27, 50 and 75-100 kDa proteins. Bands were seen in zymograms around 100 kDa.
  
  
  CONCLUSIONS
  Low serum IgA levels are associated with Blastocystis carriage. All IBS patients were more likely to demonstrate reactivity with Blastocystis proteins of 27 kDa (likely a cysteine protease), 50 and 75-95 kDa MW compared to HC. The presence of antibodies to these Blastocystis proteins in some Blastocystis-negative subjects suggests either prior exposure to Blastocystis organisms or antibody cross reactivities. The anti-proMMP-9 reaction at 50 and 75-100 kDa and the zymogram result suggest that metalloproteases may be important Blastocystis antigens.
  
  
  BACKGROUND
  Australian and New Zealand Clinical Trials registry ACTRN: 12611000918921.
authors.affiliation: School of Veterinary Science, The University of Queensland, Gatton Campus, Gatton, QLD, 4343, Australia. robynnagel@tgclinic.com.au.|Toowoomba Gastroenterology Clinic, Suite 105 Medici Medical Centre, 15 Scott St, Toowoomba, QLD, 4350, Australia. robynnagel@tgclinic.com.au.
authors.forename: Robyn
authors.initials: R
authors.lastname: Nagel
authors.affiliation: School of Veterinary Science, The University of Queensland, Gatton Campus, Gatton, QLD, 4343, Australia. rtraub@unimelb.edu.au.|Faculty of Veterinary and Agricultural Sciences, University of Melbourne, Parkville, VIC, 3052, Australia. rtraub@unimelb.edu.au.
authors.forename: Rebecca J
authors.initials: RJ
authors.lastname: Traub
authors.affiliation: Rural Clinical School, School of Medicine, The University of Queensland, Toowoomba, 4350, Australia. m.kwan@uq.edu.au.
authors.forename: Marcella M S
authors.initials: MM
authors.lastname: Kwan
authors.affiliation: School of Veterinary Science, The University of Queensland, Gatton Campus, Gatton, QLD, 4343, Australia. h.bielefeldtohmann1@uq.edu.au.|Australian Infectious Diseases Research Centre, The University of Queensland, St. Lucia, QLD, 4072, Australia. h.bielefeldtohmann1@uq.edu.au.
authors.forename: Helle
authors.initials: H
authors.lastname: Bielefeldt-Ohmann
mesh_terms: D000913:Antibodies, Protozoan / Q000097:blood*; D016844:Blastocystis / Q000276:immunology*; D016776:Blastocystis Infections / Q000453:epidemiology* / Q000276:immunology; D015153:Blotting, Western; D002353:Carrier State / Q000276:immunology / Q000469:parasitology; D016054:DNA, Protozoan / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D005243:Feces / Q000469:parasitology; D006801:Humans; D007070:Immunoglobulin A / Q000097:blood; D043183:Irritable Bowel Syndrome / Q000150:complications*; D009520:New Zealand; D016133:Polymerase Chain Reaction; D015995:Prevalence; D044967:Serum / Q000737:chemistry / Q000276:immunology
chemical_list: D000913:Antibodies, Protozoan; D016054:DNA, Protozoan; D007070:Immunoglobulin A