On Blastocystis and Animal Models

I was recently encouraged by one of my readers to do a blog post on Blastocystis and animal experimental models. This is not exactly my core competence, which probably boils down to the fact that animal models have only been scarcely used in Blastocystis research for reasons that I will try to account for below.

Animal models (mice, rats, guinea pigs) have often been used to study interactions between hosts and microbes as well as the effect of chemotherapeutic interventions. Therefore, one might assume that animal models are an obvious way of potentially establishing a link between Blastocystis and pathology. But currently, the rationale for carrying out some types of Blastocystis experiments on, say, mice or rats is limited. Why? Well, first and foremost because of at least three major issues.

1) Lack of correlation between in vitro and in vivo evidence. Experimental infections of laboratory mice (Elwakil and Hewedi, 2010) resulted in tissue invasion - something never reported in humans. Another study showed increased oxidative stress in Blastocystis infected rats (Chandramathi et al., 2010), again something not linked to human colonisation. Studies that provided evidence for induction of cytokines, contact mediated apoptosis, and barrier disruption all used axenic Blastocystis and in vitro mammalian cell cultures with no evidence that these effect occur in vivo.

2) Host specificity. Blastocystis exhibits extreme genetic diversity and multiple, genetically very different variants (species, subtypes) exist. These subtypes exhibit moderate host specificity. This means that some subtypes are common in one type of host, whereas other subtypes are common in other types of hosts. For instance, ST5 is very common in pigs, but we rarely see it in humans. ST4 is common in rodents, and in some human populations (mainly Europe it seems), but otherwise extremely uncommon. And so on. This means that some subtypes may be difficult to establish in experimental animals. It also means that any pathology detected in the animal, may not be “reproducible” in another host, - maybe due to the fact that this host has adapted to this particular subtype or even strain. Blastocystis is common in a huge variety of animals, and different animals may have adapted do different subtypes. It is not unlikely that this is due to co-evolution, and therefore it may not turn out to be a big surprise if Blastocystis per se is not usually directly associated with disease. It may still be so, however, that for humans, some subtypes or strains may be associated with disease, preliminary data point in this direction.

3) Study design. Another issue is the use of appropriate controls – for example, experimental infection of animals with Blastocystis from cultures growing with bacteria need to have the appropriate controls - namely infection with the accompanying bacterial flora alone – before it can be concluded that Blastocystis is responsible for any effects seen. It is extremely difficult to axenise (i.e. make sterile) Blastocystis strains, so they will always be accompanied by some bacterial species. Hence, any effect noticed after challenge with a Blastocystis strain will be difficult to interpret, - is it due to Blastocystis or to accompanying bacterial strains? (If you want to see what Blastocystis look like in culture, go to my previous blog post here.)

So, results from scientific studies using animal experimental models should be interpreted cautiously. In vitro experimental models using enterocyte mono-layers for instance may constitute a more attractive alternative, but the problems of using xenic (i.e. unsterile) strains are evident also here. A great challenge ahead is the development of a standardised method for axenising (sterilising) strains… so far, such a method does not exist.

Our French colleagues recently published the genome of Blastocystis sp. ST7. Functional genomic analysis is key to understanding the extent to which Blastocystis is capable of exerting any direct pathological effect, and will assist us in studying the potential pathogenicity of Blastocystis in the absence of a suitable animal model. Indirect pathological effects may be more difficult to identify and probably require studies of the interaction between the host, the parasite and the rest of the gut microbiota (bacteria). Given our recent technological advances, I believe that a pathway to knowledge lies in the study of Blastocystis in an ecological context. I think that we should get an understanding of: 1) Who are colonized with Blastocystis, 2) From where do we get it, 3) For how long do we have the parasite, and do we establish symptoms in the very beginning, only to adapt to the presence of the parasite later on, 4) does Blastocystis require a particular flora to establish (and are there differences between subtypes (in humans and animals)), 5) could Blastocystis be seen as a proxy for a given gut microbiota (biomarker), and/or does Blastocystis select for a given microbiota phenotype (metatranscriptomic analysis of the intestinal flora accompanying Blastocystis might be useful to study how the bacteria “behave” (i.e. gene expression) in the presence/absence of Blastocystis), 6) are any Blastocystis-induced symptoms related to parasite abundance, etc.; this can be explored in rough detail by using real-time PCR, of which two have been published.

So, while animal models may not be immediately suitable in our quest to study Blastocystis pathogenicity, our “omics” methodologies and data analyses may sooner than we know help us answer many of the questions that we have been pondering for decades.

Having said that, I think that for instance a pig experimental model might be useful in terms of studying the effect of chemotherapeutic intervention. Obvious studies include those aiming to identify drugs capable of eradicating Blastocystis, but it could also be interesting to study the structure and function (gene expression profiling) of the accompanying microbiota before and after intervention.
Since pig feed often contains a range of antibiotics, it could be interesting to test whether pigs on diets +/- antibiotics differ in terms of Blastocystis colonisation... a recent PNAS paper demonstrates a shift in the structure and function of the microbiome in medicated pigs compared to pigs fed a diet void of antibiotics.

Further reading:

Chandramathi S, Suresh KG, Mahmood AA, & Kuppusamy UR (2010). Urinary hyaluronidase activity in rats infected with Blastocystis hominis--evidence for invasion? Parasitology research, 106 (6), 1459-63 PMID: 20358228

Elwakil HS, & Hewedi IH (2010). Pathogenic potential of Blastocystis hominis in laboratory mice. Parasitology research, 107 (3), 685-9 PMID: 20499092

Hussein EM, Hussein AM, Eida MM, & Atwa MM (2008). Pathophysiological variability of different genotypes of human Blastocystis hominis Egyptian isolates in experimentally infected rats. Parasitology research, 102 (5), 853-60 PMID: 18193282 

Iguchi A, Ebisu A, Nagata S, Saitou Y, Yoshikawa H, Iwatani S, & Kimata I (2007). Infectivity of different genotypes of human Blastocystis hominis isolates in chickens and rats. Parasitology international, 56 (2), 107-12 PMID: 17251054

Looft T, Johnson TA, Allen HK, Bayles DO, Alt DP, Stedtfeld RD, Sul WJ, Stedtfeld TM, Chai B, Cole JR, Hashsham SA, Tiedje JM, & Stanton TB (2012). In-feed antibiotic effects on the swine intestinal microbiome. Proceedings of the National Academy of Sciences of the United States of America, 109 (5), 1691-6 PMID: 22307632

Scanlan PD (2012). Blastocystis: past pitfalls and future perspectives. Trends in parasitology PMID: 22738855

Stensvold CR, Alfellani MA, Nørskov-Lauritsen S, Prip K, Victory EL, Maddox C, Nielsen HV, & Clark CG (2009). Subtype distribution of Blastocystis isolates from synanthropic and zoo animals and identification of a new subtype. International journal for parasitology, 39 (4), 473-9 PMID: 18755193

Stensvold CR (2012). Thinking Blastocystis out of the box. Trends in parasitology PMID: 22704911

Yan Y, Su S, Ye J, Lai X, Lai R, Liao H, Chen G, Zhang R, Hou Z, & Luo X (2007). Blastocystis sp. subtype 5: a possibly zoonotic genotype. Parasitology research, 101 (6), 1527-32 PMID: 17665214