Title: Gene expression in early-weaned piglets in relation to susceptibility to enterotoxigenic Escherichia coli F4ab/ac.
Other Titles: Genexpressie in pas gespeende biggen in relatie tot hun gevoeligheid voor enterotoxigene Escherichia coli F4ab/ac.
Authors: Schroyen, Martine
Issue Date: 21-May-2012
Abstract: In the pig farming industry, intestinal diseases are known to be one of the major problems in nursing piglets. This causes a lot of losses, not only due to a higher mortality but also to giving the necessary extra attention to sick piglets, their higher feed conversion ratio and subsequently the longer period of housing them.One well known intestinal disease in piglets is post-weaning diarrhoea. The problem occurs at the moment of weaning because of the immunological status of the piglets at that time in life. While still with their mother, the piglets can be passively protected against micropathogens by maternal antibodies, which they obtain through the sow's milk. Once weaned, passive protection is lost. The pathogen causing post-weaning diarrhoea is enterotoxigenic Escherichia coli (ETEC). ETEC colonises the small intestine by attaching itself to it with its fimbriae, such as F4 (ETEC-F4) and F18 (ETEC-F18). Once ETEC is attached to the small intestine of the piglet, it can multiply itself and produce toxins. One of the most important toxins is the heat labile toxin (LT). This toxin binds to GM1, which is a ganglioside on the villi of the small intestine of the pig. By binding of LT to GM1, a cascade of events will start which will lead to the opening of Cl- channels. An excessive amount of water will accumulate in the small intestine of the piglet and it will suffer from diarrhoea.The inheritance for ETEC-F18 susceptibility is under control of one single locus on Sus scrofa chromosome 6 (SSC6) consisting of two alleles with susceptibility dominating resistance. There is substantial evidence for a genetic susceptibility to ETEC-F4 as well, since not all pigs suffer from diarrhoea after an ETEC-F4 infection. A region on SSC13, between markers Sw207 and S0075, has been found to be in close linkage to the susceptibility of piglets for ETEC-F4ab,ac, the two most important variants of ETEC-F4 (Edfors-Lilja et al., 1995). Potential candidate genes on SSC13 have been examined in many studies and although some polymorphisms were found to be in linkage disequilibrium with the phenotype, the causative mutation has not been found yet. In this study, rather than looking at potential polymorphisms in positional candidate genes on SSC13, the focus was on finding gene expression differences between ETEC-F4ab,ac susceptible (have a receptor for F4ab,ac, also referred to as F4ab,acR+) and ETEC-F4ab,acR- (do not have a receptor for F4ab,ac, referred to as F4ab,acR-) resistant piglets in those candidate genes in the hope to find a mechanism that could explain the difference in phenotype.Because gene expression is a snapshot in time, gene expression was measured during an ETEC-F4 outbreak. First of all, distinction was made between F4ab,acR+ and F4ab,acR- by means of the brush border adhesion assay. Then, before performing quantitative reverse transcription PCR (qRT-PCR) experiments to measure gene expression in both groups, it was necessary to obtain a good normalisation strategy. In order to do so, several housekeeping genes were checked for every experiment, and the most stable ones were selected. Expresssion levels of genes of interest could then be normalised with the geometric mean of the expression levels of those stable housekeeping genes. The next step was to examine the quality of the tissue samples. To this extent, the expression of two genes was examined: the genes coding for the intestinal fatty acid binding protein (IFABP) and the pancreatitis associated protein (PAP), also known as regenerating islet-derived 3 alpha (REG3A). The IFABP expression can be seen as a standard for the integrity of epithelium. Since IFABP expression was not stable in both experiments performed, this had implications on the results of other gene expression levels in these experiments. The expression of PAP was studied in order to obtain information about the inflammatory response of the animals after an ETEC infection. Since ETEC is a non-invasive, non-inflammatory pathogen, no differences should be seen in PAP regulation between piglets suffering from ETEC-F4ab,ac diarrhoea and piglets without diarrhoeal symptoms, and thus also between the susceptible and resistant piglets.The positional candidate genes whose expression in relation to ETEC-F4ab, ac seemed the most obvious, were the three MUC-genes, MUC4, MUC13 and MUC20. The MUC-genes translate in cell surface glycoproteins and a potential F4ab,ac receptor has previously been described as an intestinal mucintype glycoprotein (Erickson et al., 1992). Although a mutation in MUC4 is used in the Danish pig breeding programmes, this mutation is not the causative one for ETEC-F4ab,ac resistance, but it is located in close linkage with it (Rampoldi et al., 2011; Rasschaert et al., 2007). However, concerning gene expression, MUC4 expression was very low, as seen when measured with qRT-PCR or with micro-array analysis, and this in both F4ab,acR+ and F4ab,acR- piglets. Because of its low and non-differential expression, MUC4 expression was believed not to influence ETEC-F4ab,ac diarrhoea. For the other two MUC-genes, it has been established that both MUC13 and MUC20, which expression was abundantly in mid-jejunum, do neither have a gene expression profile that could explain the difference between F4ab,acR+ and F4ab,acR- piglets.Because these MUC-genes did not differ in gene expression although they were interesting genes to look for, in a positional as well as functional way, a different approach was chosen and a micro-array was set up. The micro-array revealed an upregulation of two genes, trefoil factor 1 (TFF1) and transferring receptor TFRC in piglets that were F4ab,acR+ compared with piglets that were F4ab,acR-. Because of the findings of Grange and Mouricout (1996) about the glycoprotein GP74 on the brush border membrane that belonged to the transferrin family and was able to bind ETEC-F4ab, TFRCwas a good possible candidate, the more because TFRC was mapped between the two markers on SSC13. Therefore, the expression differences of TFRC were validated with qRT-PCR as well and differences between F4ab,acR+ and F4ab,acR- piglets were even more significant with this technique. However, both TFF1 and TFRC expression was higher in F4ab,acR+ piglets without diarrhoea than in F4ab,acR+ piglets with diarrhoea. This expression difference for both genes could therefore also be seen as a kind of immunological response in F4ab,acR+ piglets to ETEC-F4-bacteria.
Publication status: published
KU Leuven publication type: TH
Appears in Collections:Division of Gene Technology (-)
Division of Livestock-Nutrition-Quality (-)

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