Blastocystis specific serum immunoglobulin in patients with irritable bowel syndrome (IBS) versus healthy controls

Abstract

BackgroundBlastocystis 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).MethodsForty 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.ResultsLevels 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.ConclusionsLow 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.Trial registrationAustralian and New Zealand Clinical Trials registry ACTRN: 12611000918921Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-015-1069-x) contains supplementary material, which is available to authorized users.

Asymptomatic Intestinal Colonization with ProtistBlastocystisIs Strongly Associated with Distinct Microbiome Ecological Patterns

“…Blastocystis has been associated with a variety of gastrointestinal symptoms ( 12 – 14 ) and inflammation, both within the gut ( 15 ) and systemically ( 16 , 17 ). Several studies have suggested that Blastocystis may play a role in the development of irritable bowel syndrome (IBS) ( 11 , 18 , 19 ). However, Blastocystis has often been described as an asymptomatic member of the normal intestinal microbiota ( 7 , 9 , 10 , 20 ) and has even been inversely associated with body mass index and Crohn’s disease ( 7 ).…”

Blastocystis sp. : Evaluation of polyclonal antibody prepared from crude protein for serological diagnosis using Rabbit serum

“…Some of the protein is also found in the human-infected Blastocystis (Mansour et al, 1995, Hanaa et al, 2016. Nagel et al (2015) reported that a wide range of proteins (from 17 kDa protein to 150 kDa protein) had been successfully been collected from Blastocystis including 27 kDa protein which is presumed to be as a cysteine protease. Meanwhile, 30 kDa protein has a similarity with cysteine protease of legumain type.…”

“…Other proteins such as 35 kDa protein is a cathepsin B and 40 kDa protein is a recombinant legumain (Nourrisson et al, 2016). 50 kDa protein is assumed to be a Matrix Metalloproteinase-9 (MMP-9), a member of zincbinding matrix metalloproteinases that degrade extracellular matrix namely gelatin (gelatinase B), collagen and elastin (Nagel et al, 2015). The rest of the proteins which are 64 kDa protein, 75 kDa protein, 90 kDa protein and 100 kDa protein, belongs to the protease family.…”

“…50 kDa protein is a matrix metalloproteinase-9 (MMP-9). This protein has a significant role in the parasite invasion and corresponds to the immune system altering interleukin (IL) 8 activity (Nagel et al, 2015).…”

Are Blastocystis species clinically relevant to humans?

“…The minor phyla also showed differences in our study; the rise in Proteobacteria was significantly greater in the IBS-P compared to N and conversely the rise in Actinobacteria greater in the IBS-N group. We have previously reported that Blastocystis carriage, but not symptoms, is associated with lower levels of serum IgA (Nagel et al 2015b). …”