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. 2019 Oct 26;9(11):422. doi: 10.1007/s13205-019-1956-5

Association of ACK1, TFRC polymorphism with diarrhoeagenic E. coli adhesion patterns and their jejunal expression profile in Indian Ghurrah pigs

Chandrakanta Rawat 1,#, Nihar Ranjan Sahoo 1,2,✉,#, Shivaji S Wagh 1, Pushpendra Kumar 1, Subodh Kumar 1, Arvind Sonwane 1, Salauddin Qureshi 3, Amit Kumar 1, Manjit Panigrahi 1
PMCID: PMC6815296  PMID: 31696027

Abstract

A total of 9 SNPs located in TFRC and ACK1 genes of SSC13q41 genomic region were examined for their association with the adhesion pattern of native Indian pigs using local isolate of diarrhoeagenic E. coli. Phenotypic evaluation of adhesion pattern of 150 pigs revealed 116 animals positive for adhesion, whereas 34 animals had non-adhesive phenotype. Among the adhesive animals, 6, 87 and 23 pigs were strongly adhesive, weakly adhesive and adhesive, respectively. PCR–RFLP study revealed 8 polymorphic SNPs with low to moderate PIC ranging from 7.39 to 37.25% and low to high heterozygosities (8–70%). The loci g.291 C > T, rs81218930 C > T, rs318751568 C > T of TFRC and g.93222 C > A g.94600 C > T of ACK1 showed significant departure from HWE. The genotypic frequencies of the SNPs as well as the haplotypes did not differ significantly (P > 0.05) across the adhesion patterns except one SNP (ACK1-g.107371 A > C). Among the g.107371 A > C genotypes observed, CA was associated with non-adhesive phenotype. Furthermore, TFRC mRNA expression levels were found to be significantly (P < 0.05) different among various adhesive phenotypes, whereas that of ACK1 was significantly (P < 0.05) different between non-adhesive and adhesive groups. The significant association of SNP (ACK1-g.107371 A > C), which was also previously reported to influence ETECF4 mediated diarrhoea susceptibility, implicates its wider application in genetic control of piglet diarrhoea. Furthermore, the up-regulation of TFRC gene expression in adhesive group supports its proposed role in activation of immune cells against E. coli and intracellular iron transport.

Electronic supplementary material

The online version of this article (10.1007/s13205-019-1956-5) contains supplementary material, which is available to authorized users.

Keywords: Native pig, E. coli, TFRC, ACK1, MAT, RFLP, qPCR

Introduction

Pig rearing is a major conventional occupation of tribal and rural masses in developing countries. Piglet diarrhoea, an important disease of economic significance accounts for about 11.5–29.5% of death in piglets worldwide (Li et al. 2007). Among the bacterial causes of diarrhoea, Escherichia coli are major pathogenic bacteria in swine. Enterotoxigenic E. coli (ETEC) with F4 fimbriae (previously known as K88) is a major cause of diarrhoea and mortality in neonatal as well as newly weaned pigs (Moon et al. 1999). These bacteria attach to specific receptors on brush border of small intestine by fimbriae for colonization. They synthesize enterotoxins (heat stable/heat-labile) which trigger secretion of water and electrolytes into lumen of small intestine ultimately leading to diarrhoea (Van den Broeck et al. 2000; Vu Khac et al. 2006). The adhesion of bacteria to the epithelial cells of small intestine, an essential prerequisite to cause diarrhoea is genetically controlled and inherited as a dominant character (Bijlsma et al. 1982). These receptors for adhesion are not present in every pig and their absence causes host resistance to specific E. coli strain mediated diarrhoea (Sellwood et al. 1975). The susceptible and resistant animals can be determined using an in vitro microscopic adhesion test (MAT) under unanimous standard (Baker et al. 1997). The MAT-based phenotyping and association studies allowed detection of genetic markers or candidate genes for genetic selection (Meijerink et al. 2000). In past few decades, linkage studies have shown that the loci encoding receptor for two frequent variants (F4ab and F4ac) were mapped to the 13th chromosome of the pig (Sus scrofa 13, SSC13) (Guerin et al. 1993). The target region was further refined to be located between two microsatellite loci (Sw207 and S0075) on SSC13 with fine mapping, which revealed interesting candidate genes (Edfors-Lilja et al. 1995; Jorgensen et al. 2004; Ren et al. 2009; Sinha et al. 2019). These putative positional candidate genes reported so far are mostly mucin genes (MUC4, MUC13, and MUC20), transferrin receptor (TFRC gene) and ACK1 gene [activated Cdc42 (cell division cycle 42) associated tyrosine kinase 1]. Several linkage and few expression studies reported so far on candidate genes failed to find the causative mutation for ETEC-F4ab/ac susceptibility (Schroyen et al. 2012). On the other hand, all the Escherichia coli-mediated piglet diarrhoea incidences are not caused by ETECF4, nor ETECF4 have equal prevalence all over the world. Therefore, how these SNPs (those are associated with ETEC susceptibility) behave with respect to local isolate of diarrhoeagenic E. coli-based MAT is lacking, which needs to be essentially explored for their efficient use in genetic selection program. Our previous report on the association of MUC 13 polymorphism with respect to adhesion pattern of local diarrhoeagenic non-ETEC E. coli isolate (Sinha et al. 2018a, b) encouraged us to explore the status of association with respect to the adjacent SNPs located in SSC13q41 genomic region.

Neonatal diarrhoea is one of the most common causes of morbidity and mortality responsible for heavy economic losses in Indian pig population (Shome et al. 2005). However, the literature on the genetic status of fimbrial receptors in Indian pig population controlling the adhesion pattern are scanty which would otherwise help to enhance genetic resistance against E. coli-mediated piglet diarrhoea. The Transferrin receptor (TFRC) and activated cell division cycle 42 associated tyrosine kinase 1 (ACK1) genes were assigned to SSC13q41 by radiation hybrid mapping (Van Poucke et al. 2001) and were hence considered as positional candidate genes for ETEC F4ab/ac receptor (Python et al. 2005). We aimed to explore the probable role of TFRC and ACK1 genes (i.e., to find out genetic polymorphism in these genes, ascertain their association with adhesion patterns using local diarrhoeagenic isolate and their expression profiling in different MAT phenotypes) with respect to genetic resistance against E. coli-mediated diarrhoea in Indian Ghurrah pigs.

Materials and methods

Experimental animals and tissue collection

A total of 150 desi pigs slaughtered in Bareilly City, UP were screened for E. coli adhesion pattern. Jejunum tissue samples were collected within 30 min of slaughter and brought to the laboratory in ice maintaining sterile condition. After cleaning with cold PBS, a small part (250 mg) was kept in 1 ml RNA later (Sigma-Aldrich) solution, another part (500 mg) was kept for DNA isolation at − 20 °C and about 2 cm of sample was kept at 4 °C for MAT phenotyping on the same day. Six animals of similar age (± 1 month) with different adhesion groups were subjected to jejunal expression profiling of TFRC and ACK1 genes.

Diarrhoeagenic E. coli isolation, ETEC typing, biochemical and molecular characterization

The bacterial strain (E. coli) used for adhesion assay was isolated from the diarrhoeic piglets of swine production farm of ICAR-Indian Veterinary Research Institute, Bareilly, India. They were characterized biochemically along with amplification and sequencing of partial 16S ribosomal RNA gene (gb|KJ810542) before using for adhesion test. However, while subjected to PCR-based fimbrial typing for ETECF4, they were found to be negative (Sinha et al. 2018a, b). This ETECF4 negative diarrhoeagenic E. coli isolate was cultured in BHI agar plate for overnight at 37 °C and a single colony was incubated in LB broth medium (Trypton, Yeast extraction, NaCl, pH 7.0–7.2) at 37 °C for 16–18 h at 180 rpm. Once the final OD reached 1.0 at 520 nm, the culture was used for MAT on the same day.

Phenotyping by microscopic adhesion test

The adhesion phenotypes of examined pigs were determined using MAT as described by Li et al. (2007) to explore the patterns and their frequencies in the population. Briefly, for isolation of intestinal brush borders, a piece of mid jejunum (approx. 2 cm) about 1 m proximal to the ileo-caecal valve was excised from small intestine and cut open along longitudinal axis and washed with cold PBS containing 0.1 M EDTA to make free of contents. Subsequently, the sample was rinsed gently in cold hypotonic EDTA solution. Epithelial cells were collected in cold hypotonic EDTA solution by scraping mucosal surface of jejunum with edge of a glass microscopic slide. The enterocytes were homogenized and filtered through a clean muslin cloth. The filtrate was centrifuged at 3500 rpm for 10 min and re-suspended in 5–6 ml cold PBS. The brush border suspension was added with 100 µl Gentamicin sulfate (1 mg/ml) and sodium azide (3 mM) to make a final concentration of 1 × 106 cells/ml. An equal volume of brush border cell suspension was mixed with fresh bacterial suspension (100 µl each) in a 6 ml tissue culture plate for each pig and incubated for 30 min at 37 °C at 200 rpm. A drop of the mixed suspension was examined for the adhesion pattern using a light microscope under 40 × objective. A single epithelial cell was considered adhesive when more than five (> 5) bacteria were found adhering to its brush border. Twenty well separated and intact enterocytes were checked from each of the animals. The samples were classified as strongly adhesive, adhesive, weakly adhesive and non-adhesive groups for further analysis as per Li et al. (2007). Out of 20 cells, strongly adhesive phenotype has at least 16 adhesive cells, whereas adhesive, weakly adhesive and non-adhesive phenotypes have 2–15, 1–2 and 0 adhesive epithelial cells, respectively, attached to the brush border.

Selection of SNPs and PCR–RFLP design

A total of 9 SNPs were selected based on the previous reports of Jacobsen et al. (2009) which restricted candidate region to 1.5 Mb covering several candidate genes including TFRC and ACK1. Out of 6 SNPs located in TFRC gene, two (TFRC-291 C > T and g.194965 T > C) were previously reported (Jacobsen et al. 2009; Wang et al. 2007). Four more SNPs (rs81218930 C > T, rs319005696 C > G, rs319745157 C > T and rs318751568 C > T) located in proximity were selected from dbSNP for the study. The oligo nucleotide primers flanking SNPs were designed using online software Primer3 plus tool (http://primer3plus.com/cgi-bin/dev/primer3plus.cgi) (Table S1). Likewise, already reported SNPs (g.93222 C > A, g.94600 C > T and g.107371 A > C) located at ACK1gene were targeted. The primers reported by Jacobsen et al. (2009) were used for amplification. Suitable restriction enzymes were selected for each SNP loci with the help of NEB cutter V2.0 online software (www.tools.neb.com/NEBcutter2).

Genomic DNA extraction and mass genotyping

Genomic DNA was isolated from muscle tissue (500 mg) samples by Phenol: Chloroform: Isoamyl alcohol extraction method as described by Sambrook and Russell (2001). The quality and integrity were checked by horizontal submarine agarose gel (0.8%) electrophoresis. The purity and concentration were assessed by a Nanodrop spectrophotometer (ND 1000). Samples having OD ratio (260 nm/280 nm) 1.7–1.9 were used for the further experiment with final concentration of 50 ng/µl. PCR was carried out with thermal cycler (Veriti 96 well thermal cycler Applied Biosystems) using the conditions as initial denaturation at 95 °C (5 min) and 35 cycles of denaturation at 94 °C (45 s), annealing at AT (30 s), extension at 72 °C (30 s) and a final extension at 72 °C (10 min). The digestion of PCR products was carried out with required time and temperature as provided in a kit of each restriction enzyme (Table S1). The digested PCR products were subjected to electrophoresis in 3–3.5% w/v agarose gel (low EEO) for 1.45 h at 90 V to reveal a particular pattern or RFLP genotype depending on the presence and absence of RE sites.

Expression profiling using real-time PCR

Total RNA was isolated from jejunum tissue using Trizol reagent (Thermo Scientific, USA) and chloroform (Sambrook and Russell 2001). The RNA was precipitated using isopropanol and washed with 70% ethanol. The quality and quantity were checked by nanodrop spectrophotometer and denaturing agarose gel electrophoresis. Reverse transcription was carried out using Revert Aid H Minus First Strand cDNA Synthesis Kit (Thermo Scientific, USA). Briefly, total RNA of 1 μg per reaction was dissolved in nuclease-free water with addition of oligo (dT)18 primer to make the final volume of 12 μl in a 0.5 ml microcentrifuge tube. The reaction components (5 × reaction buffer; RiboLock RNase Inhibitor; 10 mM dNTP Mix and RevertAid H Minus M-MuLV Reverse Transcriptase) were added to the tube and incubated at 42 °C and 70 °C for 60 min and 5 min, respectively, and finally at 4 °C forever. The cDNA was checked by PCR with porcine GAPDH primers (Nygard et al. 2007) to yield 90 bp amplicon. The expression profiling of target genes were carried out by quantitative Real-Time PCR (Applied Biosystems®, USA) using gene-specific primers (Table S1) and Fast SYBR Green qPCR Master mix. The reaction mix (20 µl) was prepared by adding 10 µl of the 2 × Master mix, 0.5 µl of Forward and Reverse Primer (10 pmol final concentration), 1 µl of cDNA (approx. 20 ng) and Nuclease-free water to make a final volume up to 20 µl. A qPCR amplification program was used (One cycle of Hot start PCR at 95 °C for 20 s followed by 40 cycles of denaturation at 95 °C for 3 s and annealing/extension at 58–60 °C for 30 s). The cycle threshold (Ct) values, as well as amplification plot for all three conditions (non-adhesive, weak adhesive, adhesive (merged adhesive and strong adhesive) were measured for the genes of interest (TFRC and ACK1) against an endogenous control gene (GAPDH) using the method described by Livak and Schmittgen (2001).

Statistical analysis

The association of SNPs with adhesion patterns was found out using logistic model of SAS 9.3 software (SAS Systems for Windows Version 9.00, SAS Institute Inc., Cary, NC, USA). The univariate analysis of logistic regression considered the adhesion status as a categorical response variable (adhesive, weakly adhesive and non-adhesive). The strongly adhesive pigs were clubbed with adhesive phenotype to form adhesive group positive for complete attachment (susceptibility) for all statistical purposes. The odds ratios (OR) with 95% CIs were calculated using PROC LOGISTIC procedure. The PROC ALLELE procedure of SAS 9.3 was used for testing of HWE using the Pearson Chi-square test to assess goodness of fit, estimation of PIC and heterozygosity of SNPs markers as well as linkage disequilibrium (LD) between two SNP loci. The haplotype analysis of polymorphic SNPs was done using HAPLOTYPE ANALYSIS software (Eliades and Eliades 2009) to find out the distribution of haplotypes across various adhesion patterns. The statistical significance of differences (P < 0.05) in mRNA expressions of the examined factors was assessed using one-way ANOVA with Tukey’s Multiple Comparison Test as post hoc to determine the significant differences between dCT values of the analyzed groups.

Results and discussion

Microscopic adhesion test

Out of 150 pigs, 34, 87, 23 and 6 animals were classified as non-adhesive, adhesive, weakly adhesive and strongly adhesive, respectively (Fig. S1). The effect of sex and age was found to have non-significant (P < 0.05) effects on distribution of adhesion patterns. The MAT-based phenotyping revealed that adherent bacteria were observed in the brush border of small intestines and the degree was quantified for the classification of animals. We followed the classification described by Li et al. (2007) and observed four adhesion phenotypes in sampled pigs. A fraction of 22.7% was found to be negative (i.e., non-adhesive) for adhesion. Among the adhesion positive samples, 58.0, 15.3 and 4.0% pigs were adhesive, weakly adhesive and strongly adhesive, respectively. The relatively higher proportion of adhesive pattern in our study could be either due to the small population size or use of non-ETECF4 pathotype for MAT. The incidence of about one-fourth non-adhesive pattern in native pigs indicated that enough starting material (animals with non-adhesive phenotypes) was available for breeding against E. coli-mediated diarrhoea susceptibility. Several authors (Rita et al. 1994; Yan et al. 2009) have reported breed differences in adhesion patterns in Chinese and Tibetan pig breeds. As the pigs under experimentation share similar geographical location with Chinese and Tibetan pigs the 22.7% non-adhesive phenotype was justified. While there is consensus over the correlation of adhesion to occurrence to diarrhoea in strongly adhesive and adhesive phenotypes, some authors have a different opinion on weakly adhesive phenotype. Sellwood (1980) reported that the weakly adhesive piglets did not develop diarrhoea when inoculated with F4ac variant, whereas Billey et al. (1998) opined weakly adhesive phenotype to be a false appearance. In our study for analysis purpose, we merged adhesive and strong adhesive to be in one group for all practical statistical purpose to indicate the actual fraction of the sample which have been attached to a quantity beyond threshold universally accepted to be capable to cause diarrhoea.

Genetic polymorphism and population parameters

Genotyping of 150 animals for 9 SNPs revealed one monomorphic SNP (TFRC-g.194965 T > C). Two SNPs (TFRC—rs319745157 C > T and ACK1-g.93222 C > A) yielded 2 genotypes each and other 6 had 3 genotypes each (Figs. S2, S3). The marker PICs of the SNPs were ranged from 7.39 (TFRC—rs319745157 C > T) to 37.25% (ACK1-g.94600 C > T) loci, which indicated that they were not much informative owing to their values < 50%. On the other hand, heterozygosities ranged from 8.0 (TFRC—rs319745157 C > T) to 70.0% (ACK1-g.94600 C > T). The population revealed the loci TFRC-g.291 C > T, TFRC—rs81218930 C > T, TFRC—rs318751568 C > T, ACK1-g.93222 C > A and ACK1-g.94600 C > T showed significant departure from HWE (Table 1). The linkage disequilibrium (LD) of different loci tested using χ2 probabilities revealed loci g.291 C > T (TFRC) and g.107371 A > C (ACK1), rs81218930 C > T (TFRC) and g.107371 A > C (ACK1), g.93222 C > A (ACK1) and g.107371 A > C (ACK1) were significantly (P < 0.05) associated with one another. The study of population parameters indicated that PIC values were < 0.5 but > 0.25 for SNPs rs81218930 C > T, rs319005696 C > G, rs318751568 C > T of TFRC and g.94600 C > T of ACK1genes indicating their moderately informative nature. However, PIC values of ACK1-g.107371 A > C, TFRC-rs319745157 C > T, ACK1-g.93222 C > A and TFRC-g.291 C > T were < 0.25. The observed heterozygosities (measure for assessment of genetic diversity) ranged from 8.0 to 70.0% with 6 loci having values ≥ 30% which can be used in genetic selection. Takezaki and Nei (1996) reported a marker to be useful for measuring genetic variation should have an average heterozygosity of between 0.3 and 0.8. The haplotype analysis revealed a total of 54 haplotypes out of 8 polymorphic SNPs distributed with different frequencies (Table 2; Fig. S4). Out of these, only 15 haplotypes were observed to have frequencies more than 1%.

Table 1.

Population genetic variability of TFRC and ACK1 gene SNPs in native Indian pigs

Locus N Alleles PIC Ht AD HWE (χ2-probability)
TFRC-g.291 C > T* 150 2 0.22 0.18 0.26 0.0003
TFRC-rs81218930 C > T* 150 2 0.36 0.67 0.46 < 0.0001
TFRC-rs319005696 C > G 150 2 0.36 0.42 0.46 0.2991
TFRC-rs319745157 C > T 150 2 0.07 0.08 0.08 0.6098
TFRC-rs318751568 C > T* 150 2 0.33 0.53 0.41 0.0002
ACK1-g.93222 C > A* 150 2 0.27 0.41 0.33 0.0014
ACK1-g.94600 C > T* 150 2 0.37 0.70 0.50 < 0.0001
ACK1-g.107371 A > C 150 2 0.27 0.30 0.32 0.5357
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*Indicates significant at P < 0.05

Table 2.

Haplotype frequency distribution of SNPs in various adhesion phenotypes

Haplotype code Adhesive Weak adhesive Non-adhesive Haplotype code Adhesive Weak adhesive Non-adhesive
haplo-1 0.01 0.00 0.00 haplo-28 0.01 0.00 0.00
haplo-2 0.01 0.02 0.00 haplo-29 0.01 0.00 0.00
haplo-3 0.01 0.00 0.00 haplo-30 0.01 0.00 0.01
haplo-4 0.00 0.00 0.04 haplo-31 0.00 0.00 0.01
haplo-5 0.01 0.00 0.01 haplo-32 0.03 0.09 0.01
haplo-6 0.01 0.00 0.00 haplo-33 0.08 0.09 0.09
haplo-7 0.01 0.00 0.01 haplo-34 0.00 0.00 0.01
haplo-8 0.00 0.00 0.01 haplo-35 0.00 0.00 0.01
haplo-9 0.01 0.00 0.01 haplo-36 0.01 0.00 0.01
haplo-10 0.02 0.00 0.01 haplo-37 0.02 0.02 0.01
haplo-11 0.01 0.00 0.00 haplo-38 0.01 0.00 0.07
haplo-12 0.00 0.02 0.00 haplo-39 0.00 0.02 0.00
haplo-13 0.01 0.00 0.00 haplo-40 0.02 0.02 0.00
haplo-14 0.01 0.00 0.00 haplo-41 0.06 0.04 0.03
haplo-15 0.01 0.00 0.00 haplo-42 0.03 0.00 0.00
haplo-16 0.01 0.00 0.01 haplo-43 0.01 0.00 0.00
haplo-17 0.00 0.00 0.01 haplo-44 0.00 0.04 0.00
haplo-18 0.02 0.00 0.00 haplo-45 0.00 0.02 0.01
haplo-19 0.01 0.00 0.03 haplo-46 0.01 0.00 0.00
haplo-20 0.00 0.02 0.00 haplo-47 0.03 0.04 0.04
haplo-21 0.04 0.00 0.03 haplo-48 0.08 0.07 0.06
haplo-22 0.02 0.00 0.00 haplo-49 0.04 0.04 0.01
haplo-23 0.04 0.04 0.01 haplo-50 0.00 0.00 0.01
haplo-24 0.00 0.02 0.00 haplo-51 0.01 0.00 0.00
haplo-25 0.01 0.02 0.00 haplo-52 0.02 0.00 0.00
haplo-26 0.02 0.00 0.03 haplo-53 0.30 0.35 0.29
haplo-27 0.00 0.00 0.01 haplo-54 0.01 0.00 0.00
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Association of genetic variants with adhesion patterns

The study of genetic resistance to ETECF4 mediated piglet diarrhoea so far reported that there are three types of proteins (i.e., mucin-type sialoglycoproteins, intestinal neutral glycosphingolipid and 74-kDa transferring glycoprotein) which are suggested to be putative ETECF4 fimbrial receptors (Van den Broeck et al. 2000; Schroyen et al. 2012). These putative receptor genes were mapped to SSC13q41 genomic region, same as location of our target genes (TFRC and ACK1). Furthermore, the biochemical nature of receptor protein strongly supports these genes to be candidate genes (Python et al. 2002). Several authors have also reported various SNPs on TFRC (DQ520947-g291 C > T by Wang et al. 2007; CU695181.9-g.19750 G > T by Jacobsen et al. 2009; CU695181.9-g.19750 G > T by Jacobsen et al. 2011) and ACK1 (CU914410.14-g.93765 G > -, g.94600 C > T, g.107371 A > C, g.108013 T > C, g.113132 A > G, g.114703 A > G and g.116847 A > G by Jacobsen et al. 2009) genes to be associated with ETECF4 adhesion pattern. However, as there are diarrhoeagenic pathotypes other than ETECF4 which can cause diarrhoea, the role of these SNPs or genomic region on the adhesion pattern beyond ETECF4 remains unsolved. We used the diarrhoeagenic E. coli isolate which was not ETECF4 as screened previously by PCR-based fimbrial typing (Sinha et al. 2018a) for MAT and aimed to find out the probable role of TFRC and ACK1 genes. The genotypic frequencies (Table 3) revealed that they were not differing significantly (P < 0.05) in adhesive, weakly adhesive and non-adhesive population except one (ACK1-g.107371 A > C) which was showing significant association (P < 0.05) with E. coli adhesion pattern. The CC (454 bp), CA (454 bp, 283 bp, 171 bp) and AA (283 bp, 171 bp) genotypes were observed (Fig. S2) at ACK1-g.107371 A > C locus with frequencies of 65.4, 30.0 and 4.6%, respectively, with significantly differing odds ratios (ORs) between E. coli adhesive, weak adhesive and non-adhesive population. The odds ratio of CA versus CC genotype is 0.41 that infers CA genotype (associated with non-adhesive pattern of E. coli) was found 0.41 times lesser in number in the adhesive group (Table 3) with respect to CC genotype. The locus which was significantly affecting the incidences of E. coli adhesion was having significant LD with loci TFRC-g.291 C > T, TFRC-rs81218930 C > T and ACK1-g.93222 C > A which indicated non-random association of alleles at two or more loci descended from single chromosomes (Reich 2001). In similar studies, Jacobsen et al. (2009) found 5 polymorphisms in TFRC gene, whereas Wang et al. (2007) observed a significant association between the 291 C > T polymorphism with susceptibility/resistance to ETEC F4ab/ac in a large sample of F2 animals. They found that the C allele was associated with the susceptible animals and T allele with the resistant animals and the association was more distinct for the F4ac than F4ab. Chaora (2013) also reported TFRC c.291C > T polymorphism with predominant CC genotype in pigs susceptible to ETECF4, which was in agreement with Wang et al. (2007). We found a significant association of ACK1 (g.107371 A > C) polymorphism with E. coli adhesion pattern. The CC genotype was more frequent in adhesive groups than CA genotypes, although the diarrhoeagenic E. coli we used in MAT was not ETECF4. The heterozygote (CA) was found to be associated which indicates that these might be the effect of heterosis as none of the homozygous was found to be significantly associated. Therefore, the SNP ACK1-g.107371 A > C was found to be useful for selecting for genetic resistance beyond ETECF4 mediated diarrhoea. The association study of the haplotypes of 8 SNPs with adhesion patterns failed to find any significant association with any of the adhesion phenotype group. Jacobsen et al. (2009) also found the same SNP (g.107371 A > C) in ACK1 gene was perfectly associated with the ETEC-F4ab/ac phenotype in Wild Boar x Large White, which was in agreement with our results. This finding implies that this particular SNP/gene/genomic region have got a wider role beyond ETECF4 mediated diarrhoea susceptibility (Table 4).

Table 3.

Genotype frequency distribution of porcine TFRC and ACK1 polymorphism in various adhesion phenotypes

SNP location Genotype Overall Adhesive Weakly adhesive Non-adhesive p value Odds ratios (95% CI)
Count Freq. Count Freq. Count Freq. Count Freq.
TFRC-g.291 C > T CC 114 0.76 72 0.77 20 0.86 22 0.64 0.22 0.58 (0.12–2.73)
CT 27 0.18 14 0.15 3 0.13 10 0.29 0.31 (0.06–1.652)
TT 9 0.06 7 0.07 0 0 2 0.05 1.00
TFRC-rs81218930 C > T CC 2 0.01 2 0.02 0 0 0 0 0.78 > 99.9 (< 0.001 to > 99.9)
CT 103 0.69 65 0.69 16 0.69 22 0.64 1.27 (0.63–2.54)
TT 45 0.3 26 0.27 7 0.30 12 0.35 1.00
TFRC-rs319005696 C > G CC 22 0.15 15 0.16 3 0.13 4 0.11 0.57 1.18 (0.43–3.26)
GC 63 0.42 36 0.38 11 0.47 16 0.47 0.75 (0.37–1.49)
GG 65 0.43 42 0.45 9 0.39 14 0.41 1.00
TFRC-rs319745157 C > T CC 138 0.92 83 0.89 23 1 32 0.94 0.15 1.00
CT 12 0.08 10 0.10 0 0 2 0.05 0.33 (0.07–1.51)
TFRC-rs318751568 C > T CC 67 0.45 39 0.41 12 0.52 16 0.47 0.84 < 0.001 (< 0.001 to > 999.99)
CT 80 0.53 51 0.54 11 0.47 18 0.52 < 0.001 (< 0.001 to > 999.999)
TT 3 0.02 3 0.03 0 0 0 0 1.00
ACK1-g.93222 C > A CA 62 0.41 36 0.387 11 0.478 15 0.441 0.457 0.78 (0.40–1.49)
CC 88 0.59 57 0.612 12 0.521 19 0.558 1.00
ACK1-g.94600 C > T CC 30 0.2 23 0.247 4 0.173 3 0.088 0.152 2.13 (0.57–7.96)
CT 105 0.7 61 0.655 16 0.695 28 0.823 0.85 (0.29–2.50)
TT 15 0.1 9 0.096 3 0.130 3 0.088 1.00
ACK1-g.107371 A > C* AA 7 0.05 5 0.053 0 0 2 0.058 0.041 0.94 (0.19–4.72)
CA 45 0.3 21 0.225 9 0.391 15 0.441 0.41 (0.20–0.82)
CC 98 0.65 67 0.720 14 0.608 17 0.5 1.00
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*Indicates significant at P < 0.05; The position of SNPs of TFRC gene g.291 C > T and g.194965 T > C refers to Gen Bank accession DQ520947 and CU695181.9, respectively; the position of SNPs of ACK1 gene refers to Gen bank accession no. CU914410.14

Table 4.

Allele frequency distribution of SNPs in various adhesion phenotypes

SNP location Allele Allele frequency p value Odds ratio (95% CI)
Over all Adhesive Weakly adhesive Non-adhesive
TFRC-g.291 C > T C 0.86 0.849 0.93 0.79 0.63 1.16 (0.62–2.17)
T 0.14 0.15 0.065 0.205 1.00
TFRC-rs81218930 C > T C 0.35 0.37 0.34 0.32 0.48 1.18 (0.73–1.91)
T 0.65 0.62 0.65 0.67 1.00
TFRC-rs319005696 C > G C 0.36 0.35 0.36 0.35 0.96 0.99 (0.61–1.59)
G 0.64 0.64 0.63 0.64 1.00
TFRC-rs319745157 C > T C 0.92 0.94 1 0.97 0.16 0.34 (0.079–1.53)
T 0.08 0.057 0 0.029 1.00
TFRC-rs318751568 C > T C 0.73 0.69 0.76 0.73 0.37 0.79 (0.47–1.32)
T 0.27 0.30 0.23 0.26 1.00
ACK1-g.93222 C > A C 0.78 0.806 0.760 0.779 0.525 0.836 (0.482–1.452)
A 0.22 0.193 0.239 0.220 1.00
ACK1-g.94600 C > T C 0.53 0.575 0.521 0.5 0.252 1.305 (0.828–2.057)
T 0.47 0.424 0.478 0.5 1.00
ACK1-g.107371 A > C A 0.21 0.167 0.195 0.279 0.057 0.586 (0.338–1.017)
C 0.79 0.833 0.804 0.720 1.00
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Jejunal expression profile of TFRC and ACK1 genes

The results of quantitative real-time PCR analysis revealed that porcine TFRC mRNA expression was significantly (P < 0.05) different across adhesive phenotypes with the highest level in adhesive (3.17 log2 fold), moderate level in the weakly adhesive (2.47 log2 fold) and low level in non-adhesive phenotypic group (Fig. 1a). The expression of ACK1 gene was to be found 2.24 and 1.90 log2 fold difference in weak adhesive and adhesive groups (Fig. 1b), respectively, as compared to the non-adhesive group. The difference was found to be statistically significant (P < 0.05). Trevisi et al. (2012) observed that intestinal gene expression of genes related to the innate immunity and responsive to the bacteria products was increased in susceptible pigs. However, Jacobsen et al. (2011) did not observe significant expression differences in TFRC expression in F4ab, acR + and F4ab, acR − Yorkshire piglets. On the other hand, Schroyen et al. (2013) reported an up-regulation of TFRC in the F4ab, acR + non-diarrhoeal group in comparison to the two other groups (F4R + with diarrhoea and F4R-without diarrhoea). However, the exact mechanism which led to difference in expression profile was yet to be established. Dai et al. (2017) reported differential expression of FUT1 (reported to be associated with ETECF18) gene with respect to age. We observed significant differences between ACK1 expression levels of non-adhesive and adhesive groups. Jacobsen et al. (2011) have not detected any significant difference in expression levels of the ACK1 gene between resistant and susceptible animals in the intestinal tissues (Jacobsen et al. 2011). In our present study, although ACK1-g.107371 A > C was associated with adhesion patterns, the qPCR result do not support this gene to appear as receptor gene even though it is located in SSC13q41 region. The mRNA expression profile revealed a significant difference between adhesive and non-adhesive group but expression level in weakly adhesive group fail to justify its role as a putative receptor gene. Furthermore, the structure of receptor gene was reported to encode mucin-like sialoglycoproteins, neutral glycosphingolipid or transferring glycoprotein (Van den Broeck et al. 2000; Schroyen et al. 2012) which does not match that of ACK1. ACK1 is activated by cell adhesion, which might have resulted to high level of basal auto-phosphorylation of ACK in adherent cells (Yang et al. 1999). On the other hand, TFRC mRNA expression profile revealed significant differences among adhesive, weakly adhesive and non-adhesive groups which indicates that it is important involvement in the adhesion mechanism. Furthermore, the number of adhesion increases with the increase in gene expression from non-adhesive to adhesive group. However, we failed to get any SNP from TFRC associated with the adhesion pattern. This might be due to the fact that SNPs under study do not have any role in TFRC expression. Our results may be explained as we have used a local diarrhoeagenic strain of E. coli and not ETECF4. TFRC gene codes for a carrier protein for Transferrin, necessary for iron import into the cell (Dekker et al. 2002), which might be the reason for up-regulation of mRNA expression with increased bacterial adhesion. Our results support that TFRC does not code a receptor for fimbrial adhesion, but its observed up-regulation can be a consequence of piglet’s ability to raise an effective immune response. It was also reported that an activation of immune cells can increase TFRC expression in the inflamed gut mucosa (Harel et al. 2011) which supports our interpretation.

Fig. 1.

Fig. 1

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Relative jejunal expression of TFRC and ACK1 mRNA in pigs with various types of adhesion patterns. In case TFRC, the expression levels were significantly different in each group and increases with increase in bacterial adhesion. In case of ACK1, the non-adhesive group was significantly different from other groups

Conclusion

All four different adhesion patterns were found in Indian Ghurrah pig population. The SNP ACK1-g.107371 A > C (which was previously reported to be associated with ETECF4 adhesion pattern) showed a statistically significant (P < 0.05) association with E. coli adhesion pattern using local diarrhoeagenic isolate expanding its scope in diarrhoea resistance. TFRC and ACK1 genes showed a significant difference in mRNA expression between non-adhesive and adhesive groups, which might be due to raising an immune response for protection against diarrhoea or intracellular iron transport. Although, these genes are not directly affecting resistant/susceptibility towards diarrhoea due to E. coli in pigs, they certainly could give positional genetic makers owing to their mapping locations.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (TIFF 415 kb) (415.6KB, tif)
Supplementary material 2 (TIFF 473 kb) (473KB, tif)
Supplementary material 3 (TIFF 433 kb) (433.9KB, tif)
Supplementary material 4 (TIFF 216 kb) (216.4KB, tif)
Supplementary material 5 (RTF 135 kb) (135.7KB, rtf)

Acknowledgements

The authors are thankful to the Director, IVRI, Izzatnagar, Bareilly, for providing necessary facilities to carry out this research work. Furthermore, they are thankful to owners of different slaughter houses in and around Bareilly for facilitating collection of samples.

Abbreviations

ACK1

Activated CDC42 kinase 1

AD

Allelic diversity

AICRP

All India coordinated research project

AT

Annealing temperature

BHI

Brain–heart infusion

CI

Confidence interval

dbSNP

Single-nucleotide polymorphism database

DT

Digestion temperature

EDTA

Ethylenediaminetetraacetic acid

ETECF4

F4 fimbriae of enterotoxigenic Escherichia coli

Ht

Heterozygosity

HWE

Hardy Weinberg equilibrium (χ2-probability)

LB

Lysogeny broth

LD

Linkage disequilibrium

MAT

Microscopic adhesion test

MUC13

Mucin 13

MUC20

Mucin 20

MUC4

Mucin 4

N

Number of observations

OD

Optical density

PBS

Phosphate-buffered saline

PIC

Polymorphism information content

RE

Restriction enzyme

RFLP

Restriction fragment length polymorphism

RT

Reverse transcription

SNP

Single-nucleotide polymorphism

SSC

Sus scrofa (pig)

TFRC

Transferrin receptor

Funding

The authors are thankful to Indian Council of Agricultural Research (ICAR) for providing necessary financial assistance in the form of an institute project (Grant No. IXX10577) to corresponding author as well as institute fellowship to first author during the study.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical statement

All procedures performed in studies involving animals were in accordance with the ethical standards of the institution or practice at which the studies were conducted.

Footnotes

Chandrakanta Rawat and Nihar Ranjan Sahoo have contributed equally.

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Supplementary Materials

Supplementary material 1 (TIFF 415 kb) (415.6KB, tif)
Supplementary material 2 (TIFF 473 kb) (473KB, tif)
Supplementary material 3 (TIFF 433 kb) (433.9KB, tif)
Supplementary material 4 (TIFF 216 kb) (216.4KB, tif)
Supplementary material 5 (RTF 135 kb) (135.7KB, rtf)

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