Introduction

By now veterinary research has aimed to identify new strategies for preventing and controlling diseases with non-chemotherapeutic approaches that avoid the exclusive use of drugs. Among these, the possibility of exploiting genetic resistance capabilities is of great interest, together with the adoption of global systems for improving animal welfare with a “One Health” perspective, also in the pig sector (Peltoniemi et al. 2015). Further researches are necessary for the estimation of how the combination of genomic information with phenotypic and pedigree data can predict the genetic merit of individuals, within the population, before the possible application in Marker Assisted Selection (MAS) programs (Jonas and Koning 2015).

Furthermore, in some countries there is the tendency to enhance or maintain breeding performance with minimal veterinary intervention, reducing the animal losses or the lower productivity, through the selection of genotypes resistant or “resilient” to diseases of interest, with a “candidate gene approach” or in a “genome wide discovery” (Emam et al. 2019).

Pig breeding and pig industry play a key role in both developed and developing countries, contributing, in the pre-pandemic period, to 37% of total meat production, worldwide (FAO 2013). Forecasts for the future, indicated in the FAO-OECD report on agricultural prospects for the period 2020–2029, estimate that pork food product will be one of the main drivers of the increase in meat demand in the medium-long term (FAO-OECD 2021).

Enterotoxigenic Escherichia coli (ETEC) are responsible for diarrhoea in humans as well as in farm animals. Particularly, in pig farming, one of the main causes of death in piglets, and consequently of huge economic losses, is pre- and post-weaning diarrhoea caused by ETEC, whose control strategy is based on therapies including rehydration and antibiotic administration. The overuse of antimicrobial drugs in diarrhoea treatment has led to the development of antibiotic resistance in most of the E. coli pathotypes associated to piglet intestines (Shakuntala et al. 2017). Another control strategy of neonatal ETEC infections is represented by vaccinating sows. An intramuscular vaccine is used to immunize pregnant sows by stimulating the production of maternal antibodies that protect piglets against ETEC diarrhoea during lactation thus against the more sever clinical form. The vaccines, in general administered parentally, elicit specific antibodies in the colostrum and milk of sows and provide passive immunity to piglets until weaning, therefore not protecting against Post Weaning Diarrhoea (PWD), (Ruan et al. 2011; Matias et al. 2017). In piglets, from 18 days of age, live oral vaccines containing E. coli strains, reduce both the incidence of PWD caused by E. coli in infected pigs and the fecal shedding of ETEC F4 and ETEC F18, eliciting an intestinal mucosal immune response against ETEC itself. Currently, also an oral vaccine is available on the market, consisting of non-pathogenic live E. coli strains, capable of eliciting mucosal immunity against ETEC rather than systemic immunity: this is a key factor when trying to prevent PWD with vaccines in piglets (Melkebeek et al. 2013). Although several studies show positive effects of the vaccine, in general the evaluation of the vaccination effects is often controversial. Furthermore, it should also be noted that the cost of an antibiotic treatment against E. coli is one third of the price of the vaccination. Another control strategy for piglet diarrhoea is feeding with nutritional supplements, that have been shown to be useful tools, with high variable effects, for PWD control by affecting the gut microbiota (Canibe et al. 2022).

With regard to the disease evolution mechanisms, the first step of ETEC-mediated diarrhoea consists in the attachment of the bacterial fimbrial adhesins, identified as F4 or F18, to the intestinal epithelium receptors of piglets (Luppi 2017). At the enterocyte level, the genes coding for the fimbriae receptors are expressed at different levels, therefore the adhesion is “genetically determined” showing a Mendelian inheritance (Bijlsma et al. 1982). This provides an opportunity to attempt to select pigs for resistance to ETEC-mediated diarrhoea using putative candidate genes as markers for selection. To date, also thanks to genome wide association studies (GWAS), few potential candidate genes have been proposed, namely the Mucin 4 (MUC4) gene and the alpha-fucosyltransferase-1 (FUT1) gene. Susceptibility to ETEC F4 has been associated with a single nucleotide polymorphism (SNP) located in intron 7 (DQ 848,681:g.13:8227 C > G) of the MUC4 gene (Jørgensen et al. 2004; Rampoldi et al. 2011; Luise et al. 2019). Piglets with the MUC4 G/- genotype express the F4 receptor and they are considered more susceptible to ETEC F4 infection, whereas the only MUC4 C/C genotype seems to be associated with the resistant phenotype (Jorgensen et al. 2003). On the other hand, the susceptibility for ETEC F18 infection appears to depend on the FUT1 gene, which is the candidate gene for regulation of the F18 receptor expression (ECF18R) (Meijerink et al. 1997). The g.6:54079560 A > G SNP located on the FUT1 gene, referred to rs335979375 in the last version Sus scrofa 11.1 of pig genome, has been associated with a major genetic susceptibility versus ETEC F18 infection. Also according to the literature, piglets with FUT1G/- genotypes appear susceptible to ETEC F18, while piglets with the only FUT1A/A genotype are resistant to the infection (Meijerink et al. 1997; Wang et al. 2012; Munoz et al. 2018). Therefore, on this basis, this study wants to be a “genetic survey”, with the specific aim of investigating, to the best of our knowledge, for the first time, the polymorphisms located on FUT1 and MUC4 candidate genes and their distribution in commercial pig populations reared in some Central Italy regions.

Materials and methods

Sample collection and DNA extraction

For this study, 362 diaphragmatic muscle samples of commercial hybrids pigs, coming from 49 herds, were used. The animals were bred in different farms located in Central Italy, for the most in the Umbria, Toscana and Lazio regions, with a structure capacity ranging from 70 to 7000 animals. The 20% of slaughtered pig’s diaphragms coming from each herd was collected. The detailed information of the farms is reported in the Supplementary Table 1. Tissue aliquots were taken by authorized veterinarians after slaughter during the mandatory controls planned for the pig breeding sector, in particular for trichinellosis inspections, therefore no ethical approval was required. Genomic DNA extraction was performed from one collected diaphragmatic tissue section per animal, using High Pure PCR Template Preparation Kit (Roche Life Science, Mannheim, Germany), following the manufacturer’s instructions. A negative extraction control (demineralized sterile milliQ water) was used for each DNA extraction session.

FUT-1 and MUC-4 PCR-RFLP

PCR amplifications of target genes were performed in a final volume of 25 µL containing 60–100 ng of genomic DNA. Specific primers for FUT1 and MUC 4 genes were selected from Coddens et al. (2008) and from Sterndale et al. (2019), respectively (Table 1).

Table 1 Primer sequences and details of target genes FUT1 and MUC4
Full size table

PCR amplification mixes for both genes contained: 1X GoTaq® Flexi Buffer, 1.5 mM MgCl2, 1 U GoTaq® G2 Flexi DNA Polymerase (Promega Corporation, Madison, WI, USA), 200 µM dNTPs (GE Healthcare, Buckinghamshire, England), 0.4 µM of each primer (Sigma-Aldrich, Saint Louis, MO, USA). PCR protocols were carried out with the following thermal cycling profiles: an initial denaturation step at 95 °C for 5 min followed by 35 cycles at 95 °C for 40 s, 55 °C for 40 s, 72 °C for 45 s and a final extension step at 72 °C for 10 min, for FUT1 gene, an initial denaturation step at 95 °C for 5 min followed by 35 cycles at 95 °C for 30 s, 54 °C for 30 s, 72 °C for 30 s and a final extension step at 72 °C for 10 min, for MUC4 gene. PCR reactions for FUT 1 and MUC 4 genes gave amplification products of 421 bp and 367 bp, respectively. PCR-RFLP based on the G/A mutation of FUT1 gene was used for the determination of the “F18 genotype”. The FUT1 PCR product was digested in a total volume of 20 µl with Hin6I restriction enzyme. Furthermore, to determine the “F4 genotype”, PCR-RFLP based on the C/G mutation was performed by digesting MUC4 amplicon with XbaI restriction enzyme. For FUT1 gene, two bands of 328 bp and of 93 bp visualized on UVITEC Cambridge correspond to AA resistant genotype, while three bands of 241 bp, 93 bp and 87 bp correspond to GG susceptible genotype. For MUC 4 gene, CC resistant alleles were viewed as a single band at 367 bp and GG susceptible alleles were viewed as two bands at 216 and 151 bp. All the digestion products for both genes were visualized on 3% agarose gel containing Midori Green Advanced DNA Stain. In order to confirm the digestions results, 20 PCR amplicons of both FUT1 and MUC4 genes were sequenced by Sanger approach. The sequencing reactions were performed using the BrilliantDye™ Terminator Cycle Sequencing Kit v3.1 (NimaGen BV, Nijmegen, The Netherlands) in accordance with the manufacturer’s instructions. Sequencing reactions were run in a 3500 Genetic Analyzer (Thermo Fisher Scientific). All sequences, in FASTA format, were aligned to Sus scrofa FUT1 gDNA, complete cds (GenBank Accession Number: U70883.2) and to Sus scrofa MUC4 gDNA, partial cds (GenBanck Accession Number: DQ124298.1) and then analyzed with BioEdit v7.2.5 software (Hall et al. 1999), using the ClustalW algorithm.

Results

For FUT1 gene, the three expected genotypes A/A (328 and 93 bp), A/G (328, 241, 93, 87 bp) and G/G (241, 93, 87 bp) were found after digestion of 421 bp PCR product. With regard to the MUC4 gene, after digestion of the 367 bp amplicon, three genotypes were detected, or C/C (367 bp), C/G (367, 216, 151 bp) and G/G (216, 151 bp). The allelic and genotypic frequencies of FUT1 and MUC4 are shown in Table 2. The digestion results were fully confirmed by the sequencing analysis. The prevalence of F18 + E. coli susceptibility genotypes FUT1G/G and FUT1G/A were estimated to be 49.2% (Confidence Interval (CI) 95% 43.9–54.4) and 39.5% (CI 95% 34.4–44.7), respectively. The FUT1A/A resistant genotype to F18 + E. coli had a frequency of 11.3% (CI 95% 8.3–15.1). The allelic frequencies of FUT1A and FUT1G were 0.31 and 0.69, respectively. The prevalence of MUC4C/C resistance genotype was 86.1% (CI 95% 82.1–89.5) whereas the susceptible genotypes MUC4C/G and MUC4G/G had frequencies of 13.3% (CI 95% 10.0–17.2) and 0.6% (CI 95% 0.1–2), respectively. Allele frequencies for MUC4C and MUC4G were 0.93 and 0.07, respectively (Table 2).

Table 2 Genotypic and allelic frequencies of FUT1 and MUC4 genes polymorphisms
Full size table

Discussion

Although PWD is a multifactorial disease (Eriksen et al. 2021), enterotoxigenic E. coli (ETEC) expressing the fimbriae F4 and F18 are considered as the main etiological agents (Luppi et al. 2016; Rhouma et al. 2017). An estimation of the prevalence of ETEC F4 and ETEC F18 among herds with PWD in Italy is around 46.4% and 29.8%, respectively (Luppi et al. 2016). It is relevant to know that ETEC infection can also exacerbate the intestinal microbiota dysbiosis increasing the susceptibility of weaned pigs to other enteric infectious diseases (Kim et al. 2022). Several studies focused on the weaning period that produces clear changes in the piglet’s gastrointestinal tract. Particularly, weaning transitions have shown that switching piglets from sow’s milk to a solid diet decreases the Lactobacillus spp cluster in the gut microbiome and an increase in facultative anaerobes such as Clostridium spp, Prevotella and E. coli (Pluske et al. 2018; De et al. 2019) is observed. The administration of oral E. coli vaccines, containing wild-type live avirulent strains, to weaned piglets has been proposed to induce local production of E. coli antibodies thus preventing the adhesion of pathogenic E. coli to intestinal cells. As already reported, controlling PWD diffusion requires different strategies. Besides treatments and preventive approaches, the outcomes of several studies have shown that the host FUT1 and MUC4 genes are associated to diarrhoea, playing a key role in natural genetic resistance or susceptibility to ETEC F18 and ETEC F4 infection in pigs (Munoz et al. 2018; Luise et al. 2019). Genetic investigations may contribute to a deeper understanding of host-microbial crosstalk (Luise et al. 2019). In particular, the study of Jorgensen et al., 2003 has highlighted that the MUC4 SNP is associated with piglet susceptibility for ETEC F4ac, influencing the piglet growth performance and the faecal score during the first weeks post‐weaning. Pigs with the resistant genotype (MUC4CC) remained apparently healthy or had firmer faeces than the pigs with the susceptible genotypes (Jorgensen et al. 2003).

Up to date, among the three MUC- candidate genes (MUC4, MUC13 and MUC20) the MUC4-g.8227 G > C is the most extensively studied polymorphism in relation to ETEC F4ab/ac variants susceptibility in pigs. Since this gene was found to be associated with ETEC-F4 suceptible/resistant genetic profile in different populations, the identification of the causative mutation(s) that condition and affect F4ab/ac susceptibility/resistance could lead to a selection strategy to control piglet diarrhoea. In addition, it was assumed that it could be a valid marker for MAS application even if not ubiquitously for all pig populations/breeds (Schroyen et al. 2012).

On this basis, also taking into account literature data as well as experimental and conditioned trials, in our investigation, the prevalence of the F18 + E coli susceptible genotypes FUT1GG and FUT1GA was evaluated and it was estimated to be globally equal to 89%. Conversely, the prevalence of the resistant genotype FUT1AA was 11% with an allelic frequency of FUT1A of 0.31, probably demonstrating in the studied population the absence of selection for F18 E. coli resistant pigs. Other studies suggest that the FUT1 genotype can influence the physiological conditions and the balance of the microbiota, and this influences the number of intestinal hemolytic bacteria and of Enterobacteriaceae family (Riis Poulsen et al. 2018). On the other hand, in the investigated pig population it was registered a prevalence of 86% for the MUC4CC resistant genotype, with an allelic frequency of MUC4C of 0.93. Therefore, it can be concluded that animals bred in Central Italy seem to be susceptible to F18 + E coli but, on the contrary, they represent an important resource of favorable genetic traits and markers for the breeding of pigs resistant to F4 + E. coli. Before the implementation of these markers and genetic loci in MAS programs, further studies will be necessary to confirm these preliminary outcomes on a larger swine population. The final aim is the assessment and the association of the genotype with a defined phenotype, in particular with the presence of F4 + and F18 + E. coli disease/infection state, through the direct characterization of the pathogen (for example in faeces), the gut microbiota and/or the serological status.

However, beyond the genetics of the animal, also in order to fight the antimicrobial resistance, recent evidence has highlighted the pivotal role of nutritional interventions, including modified feeding strategies and nutrient supplements, in the control of diarrheal disease caused by ETEC (Kim et al. 2022). A combination of these interventions is very probably the best and desirable strategy to follow and to adopt for a correct and profitable pig herd management.

The potential and more effective strategy based on MAS programs could be applied using genetic panels that include the recognized and robust traits of resistance to ETEC F4 and ETEC F18.

In particular, in order to select the animals in a herd for ETEC F4 or ETEC F18 resistance, it is imperative that the genotype and thus the causative mutations affecting the adhesion of fimbriae with their respective receptors in intestinal cells must be strictly evaluated and identified, as well as in this interesting survey. The main perspective is the decreasing of the spread and circulation of ETEC strains and pathotypes in pig breeding farms and then the reducing of the public health risks related in a One Health scenario.