Introduction

Under natural conditions, the piglet’s weaning process is carried out gradually with piglets getting weaned at ages ranging from 14-17 weeks [1], whereas commercially, according to pig husbandry practised in the UK, piglets are traditionally weaned at ages ranging between 3-4 weeks [2,3]. However, the early weaning of piglets may cause a series of adverse effects on growth and development, resulting in physiological disorders in weaned piglets and the occurrence of diarrhea and edema.

It is well documented in many studies that, diarrhea is believed to be one of the most frequent major leading problems in piggery industries, particularly during both pre-and post-weaning periods [4-7]. This is not unusual due to weak/poor disease resistance, gastrointestinal anomaly and environmental stress thus resulting in huge economic losses. In some situations, it causes severe morbidity as well as mortality [8-11]. This disease alone is responsible for 11.5%-29.5% of deaths in piglets. Escherichia coli, the major infectious agent causing diarrhea in swine accounts for 56.2% of the cases of piglet diarrhea and 24.7% of the deaths as a result of diarrhea. On the other hand, the Porcine Epidemic Diarrhea Virus (PEDV), a causative agent of Porcine Epidemic Diarrhea (PED), an acute and highly contagious disease of swine leads to massive dehydration with a high mortality rate in newborn piglets due to severe vomiting and diarrhea. In 2013-2014, swine producers in the US lost $900 million to $1.8 billion as a result of the PED outbreak [12].

In actual production, some measures have been taken to prevent and treat piglet diarrhea, such as feeding antibiotics, probiotics, enzyme preparations, antibacterial peptides, specific yolk antibodies and pharmacological doses of Zinc Oxide [1315]. These drugs and feed have achieved some effective additives in the prevention and treatment of piglet diarrhea, but some also have brought disadvantages to the pig industry [16]. This has made several countries reduce and/or totally ban the use of antibiotics as growth promoters [17]. In 2003, Korea announced phasing out plans for the use of antibiotics supplements in compound feed, which came into effect in May 2005 and the use of antibiotics was totally discontinued on July 1, 2011 [18]. Similarly, in January 2006, the European Union has joined the move and banned the use of antibiotics as feed additives in food animals [19]. Subsequently, the Chinese Ministry of Agriculture has also issued a series of new regulations prohibiting the use of some antibiotics in edible animals [20]. More recently, in December 2013, the United States phased out the use of non - Medical antibiotics [21]. All these cautionary measures are initiatives to mitigate the disadvantages associated with the use of antibiotics as feed additive in food animals and to ensure safeguard and wholesome livestock products are produced.

In addition, for acute diarrhea, such as diarrhea caused by the porcine epidemic diarrhea virus, there is no commercial vaccine that has been proven effective in pig farms in the United States [22,23]. In the long run, we should carry out disease resistance breeding programs, improve resistance to pathogens by genetic means, and combine immunization to control the disease of livestock, which is essentially a way to solve the root of the problem or mitigate the diseases of livestock and poultry. This review aimed at focusing on piglets’ diarrheal infections caused by enterotoxigenic E. coli (ETEC) and porcine epidemic diarrhea virus (PEDV) as well as underlying virulence factors and resistance candidate genes.

Piglet diarrhea is a typical multifactorial disease. There are a wide variety of factors that cause diarrhea to piglets, which can be divided into two categories, non-infectious and infectious factors: Non-infectious Factors such as inadequate piglet immune system, dyspepsia, malnutrition, weaning stress, etc. can lead to piglet diarrhea. Infectious Factors include viral diarrhea, bacterial diarrhea and parasitic diarrhea [24]. The viral diarrhea is mainly transmissible gastroenteritis in piglets, epidemic diarrhea and rotavirus infection. Bacterial diarrhea is commonly induced yellow scour of piglets, white scour of piglets [25-27], post-weaning diarrhea of piglets [28] and porcine edema disease [29].

In production practice, bacteria and viruses are the main causes of diarrhea in weaning piglets, including pathogenic Escherichia coli and porcine epidemic diarrhea virus [30]. They have a high fatality rate and cause a great loss to the pig industry. Enterotoxigenic Escherichia coli (ETEC) is the main pathogen causing diarrhea in newborn piglets and weaned piglets [31,32]. E. coli strains that caused severe diarrhea are usually F4 (K88), F5 (K99), F6 (987P), F41, and F18. Among these, the pathogenic Escherichia coli expressing F4 pilus antigen is the most common, and it can cause diarrhea and death in newborn piglets and earlyweaned piglets [33], while ETEC F18 mainly cause diarrhea and edema in weaned piglets [34]. Porcine epidemic diarrhea virus (PEDV), a member of the Alphacoronavirus, causes acute diarrhea, vomiting, dehydration, and high mortality in newborn piglets, resulting in a significant economic loss [35]. In the United States, PEDV has caused a sharp rise in the mortality of piglets and continued to spread throughout the United States. The first case of the disease occurred in April 2013. Only a year later (March 2014), about 50% of the 27 states in the US were found infected with PEDV. This extremely serious negative impact has forced international cooperation among countries to work together combinatorially to mitigate its impact and stop the virus from further spreading [36].

Theodore Escherich (1857-1911) a German pediatrician and a bacteriologist, in 1885 discovered a bacterium that was found in the faeces of healthy individuals which he named Bacterium coli commune (Escherich, 1885, cited in [37]. Currently, this microorganism is known as Escherichia coli (renamed after

him, in 1919). However, some E. coli strains have developed specific abilities which turn them into primary pathogens, responsible for a range of different diseases in both man and animals [38]. The enteric E. coli pathogens are further divided into six serotypes on the basis of virulence properties, such as Enterotoxigenic Escherichia coli (ETEC), Enteropathogenic Escherichia coli (EPEC), Enteroinvasive Escherichia coli (EIEC), Verotoxigenic Escherichia coli (VTEC), Enterohaemorrhagic Escherichia coli (EHEC), Enteroaggregative Escherichia coli (EaggEC) [39,40]. Enterotoxigenic E. coli (ETEC) is the major infectious agent causing diarrhea in swine, which accounts for most cases of deaths in porcine herds [12].

The most important ETEC virulence factors in diarrhea are fimbrial adhesins and enterotoxins [41]. Fimbrial Adhesins: mediate bacterial attachment and initiation of bacterial colonization on the surface of host epithelium cells. The adhesive fimbriae most frequently found in ETEC from pigs with post-weaning diarrhea (PWD) are those belonging to types F4 (heretofore known as K88) and F18 (F107, 2134P, 8813) [42,44]. Enterotoxins: play a pivotal role in the inhibition of intestinal fluid homeostasis causing fluid hypersecretion into the gut lumen thus, leading to diarrhea. The K88 (F4) fimbrial ETEC strains often produce both heat-labile (LT) and heat-stable (STb) enterotoxins with or without STa. The F18 fimbrial ETEC strains produce STa and STb heat-stable toxins, occasionally with Stx2e toxin [45]. The most frequently observed enterotoxin combinations are LT and STb, or LT, STa, and STb [46].

The main enterotoxins detected in porcine ETEC are heatlabile toxin (LT), heat-stable toxin a (STa) and heat-stable toxin b (STb). Some strains can produce both enterotoxins and a Shiga toxin, which is usually Stx2e subtype. Some authors classify these strains as ETEC rather than Shiga toxin-producing E. coli (STEC) [47]. Despite its relevance, only a few recent studies investigating the occurrence and the distribution of fimbriae and virulence factors among E. coli isolates from cases of PWD in Europe are available and in many cases, they do not differentiate between E. coli isolates recovered from PWD cases, edema disease and preweaning diarrhea [8]. For ED and/or PWD-causing E. coli, the most common adhesins are associated with fimbriae F18, F4, F5, F6 and F41, while the predominant toxins are heat-labile enterotoxin (LT), heat-sensitive enterotoxin (ST) and Shiga toxin 2e (Stx2e) [48].

F4 fimbria can exist as antigenic variants, F4ab, F4ac and F4ad, all of which allow adhesion to villous enterocytes throughout the small intestine. There are also other fimbrial structures, such as F5 (K99), F6 (987P), F41, etc., which are associated with porcine ETEC strains but not frequently associated with PWD strains [42]. In addition, F18 (F107) fimbria has been identified on porcine E. coli strains connected with edema disease [48] and post-weaning diarrhea [4,34]. The proper characterization of E. coli strains isolated either from diarrheal or edema disease cases requires the determination of both fimbrial and toxin properties. Fimbrial antigens of F4, F5, F6, F18 and F41 in the 215 isolates of coli were detected by Xiang et al. from pigs with post-weaning diarrhea in Eastern China [49]. Luppi et al. identified two different fimbrial subtypes of ETEC in one farm, F4-ETEC (F4, STa, STb, LT) and F18-ETEC (F18, STa, STb) [50]. Reports from Poland, Slovakia and Denmark suggest differences between countries in the prevalence of the main fimbrial types in E. coli isolates recovered from pigs with PWD [51].

Infections with ETEC that, express the F4ab or F4ac fimbriae, cause diarrhea and mortality in neonatal and weaned piglets, these pathogens bind with their F4ab/ac fimbriae to F4 specific receptors (F4R) on the brush border of small intestinal enterocytes, resulting in colonization of bacteria to porcine intestinal epithelial cells (Figure 1) [52].

Colonizing bacteria then release harmful enterotoxins that lead to more secretion of electrolytes into the gut lumen and stop the process of fluid homeostasis, consequently permitting water to flow into the gut lumen causing diarrhea [34]. However, some pigs are naturally resistant to ETEC, because they don’t have receptors on their epithelial cell brush borders to which the fimbriae bind [54,55], the absence of receptors causes resistance to ETEC induced diarrhea [56] and their expression is genetically determined and inherited in a dominant way following Mendelian inheritance [53-58].

Porcine epidemic diarrhea virus is an enveloped single-strand RNA (ssRNA) virus [59] that belongs to the family Coronaviridae, genus Alpha coronavirus [60-62] causes porcine epidemic diarrhea (PED), an acute and highly contagious disease of pigs, which leads to massive dehydration and high mortality rate in baby pigs due to severe vomiting and diarrhea [63,64]. Thus resulting in huge and significant economic setbacks in pig farms [65].

PEDV was first reported in 1971 in England [60] and now worldwide including the USA, Mexico, Italy, Japan, France, Belgium, Korea, Thailand and Canada [66] poses huge economic losses. Serious PED was reported in 2010 in China [59].

Swine, the only known natural host of porcine epidemic diarrhea virus (PEDV) [67], the infection in swine is facilitated by porcine aminopeptidase N (pAPN), a cellular functional receptor that binds to spike proteins of PEDV coronaviruses [68,69]. The virus then replicates in the differentiated enterocytes of the porcine small intestine thus, causing severe diarrhea [70,71]. Viral pathogens are not the same as bacterial pathogens as far as their structures are concerned but some of their properties are similar in terms of their virulence and pathogenicity. It has a 28 kb genome consisting of 7 ORFs, one 5’ UTR and one 3’ UTR with a polyadenylated tail. The seven ORFs encode four different structural proteins and three different non-structural proteins. The four structural proteins are spike (S), envelope (E), membrane (M) and nucleocapsid (N) and the three non-structural proteins are replicases 1a, 1b and ORF3 [72,73] (Figure 2).

The S protein holds a specific receptor binding site which is necessary for fusion of cell membrane and entry of virus and is an antigenic target that neutralizes antibodies [74] whereas the M protein, the most abundant protein on the surface, is coexpressed with E protein forming pseudo-particles that results in interfering genic activity [75]. The N protein is highly conserved, binds to virion RNA for providing a structural basis to the helical nucleocapsid, and is being used for early diagnosis [76]. On the other hand, the non-structural proteins, namely; replicases 1a and 1b are both multi-functional and are associated with the replication of the viral genome [77], and the ORF3 protein is thought to have an effect on virulence [78].

Adhesion of viruses to the host cells is mediated by adhesins, however, certain viruses which are enveloped depend on antigenic variation which is not recognizable by the immune system of the host thus avoiding immune defences. In any viral infection, the first step is the viral adhesion to specific receptors on the cells’ surface. This process is facilitated by adhesins that are part of the viral capsid or membrane envelope. The viral adhesins and viralspecific cell receptors interaction define the tropism (preferential targeting) of viruses for specific cells or tissues in the body [79]. Porcine epidemic diarrhea virus has a restricted tissue tropism, replicates well efficient in the epithelial cells of the porcine small intestinal villous. Porcine amino-peptidase N (pAPN) is largely expressed on the epithelial cells surface of the small intestine and has been recognized as the cellular receptor for this virus [71,8082].

Lee et al. in their study on the N-terminal region of the porcine epidemic diarrhea virus’s spike protein showed that the N-terminal region of the PEDV spike protein S1 domain is necessary for the recognition of the pAPN receptor. Thus, the entry of PEDV into the host cells commences with the binding to pAPN then followed by the invasion of the virus into target cells through direct membrane fusion, subsequently, the viral genome is released into the cytosol after un-coating thus marking the process of genome replication [83]. However, contrary to the aforementioned evidence for pAPN acting as a receptor for PEDV infection, Li et al. [84] and Shirato et al. [85] suggested that pAPN is not required for PEDV infection and therefore, is not the cellular receptor for PEDV. Interestingly, Shirato et al. [85] further suggested that, albeit pAPN is not the cellular receptor for PEDV, its activity promotes PEDV infection.

Having described the two pathogens (ETEC and PEDV), their virulent factors and pathogenicity, we need to throw some light on piglets’ immunity because the disease can never be discussed in isolation from

immunity. The newborn piglet lacks protective immunity against diseases, thus, it largely depends on the colostrum intake from sow’s milk to obtain passive immunity from its mother, which in turn lasts for 10-14 days after birth [86,87].

Then, once the age of the piglet reaches 21-28 days, its own immune system will start to develop, therefore piglets weaned at the ages of 14-28 days are vulnerable to getting infected because their immunity to fight disease infections is at its lowest level [88]. At this juncture, any contact with causative agents can have very serious consequences [88-90] (Figure 3).

The immune system in the pig is a complex network and is responsible for recognizing as well as repelling pathogens, thereby helping in providing protection against disease infection. The immune system fights against infections in three different ways [91] as follows:

Innate Immunity: The genetic actions of innate immunity stimulate adaptive immunity, once the innate immunity identifies a foreign organism (body), the circulating T-cells will interact with a foreign antigen, they can either kill infected cells directly or they can return to lymph tissue to incite (stimulate) a B cell response which in turn will produce the antibody that can fight the infection. The innate immune system is the first defence barrier to infection in pigs. Passive Immunity: Due to the nature of epitheliochorial of the placenta in the porcine, it is necessary for the newborn baby pig to acquire maternal immune-globulins from sow’s milk colostrum to obtain passive immune to provide protection against infections until its immune system is fully developed [91,92]. Adaptive Immunity: Adaptive immunity is a defence system of pigs, which is built on specific cellular targeting, it takes up to 96 hours after infection to develop its weaponry (antibodies) and is far more effective due to its accuracy (precision). The adaptive immunity system reacts when exposed to pathogenic organisms and vaccine antigens, thereby providing long-term immunity against future infection by similar invading pathogens and it functions throughout life [91].

Genome assembly of the pig (Sscrofa10.2) comprises 2.60 Gigabases (Gb) assigned to 18 autosomes and X and Y chromosomes with a further 212 megabases (Mb) in unplaced scaffolds [99] (Table 1).

The use of genomics tools, such as genetic and physical maps, sequencing and gene expression profiles studies provided powerful tools to uncover genetic markers and genes, which can be used to improve disease resistance. Equipped with the knowledge of the genetic basis of resistance to diseases is very important in improving animal well-being by the use of conventional molecular breeding methods [100]. Previous studies revealed that resistance to infection of certain pathogens is heritable and the identification of indicator traits that can be used in breeding programs is of paramount importance in time to come in the pig industry. EdforsLilja et al. showed that heritability estimates of responses to E. coli antigens range from 0.29 to 0.45 [100].

Estimation and application of genetic parameters on the basis of phenotypic records are one of the big challenges in the selection process for resistance to disease in the pig. Fewer genes have been identified to control disease in the pig. A typical example is the presence or absence of the receptor of F4, a cell surface antigen on some Escherichia coli, which can cause diarrhea in the pig [34,101].

A further example is piglets’ genetic resistance to the causative agent of neonatal diarrhea. A receptor found in the intestinal tract to which a certain strain of E. coli adheres is too genetically determined, whereas, a commercially available DR2 gene marker test makes possible routinely identification of genetically resistant individuals and a DR1 gene marker test further identifies those individuals which are genetically susceptible to F18 E. coli. Usage of these tests in breeding practices has significantly improved genetic resistance in those populations where it has been applied [100]. Additional examples explaining how genetically resistant individuals are being favored during selection include the usage of a commercial test for the porcine syndrome (PSS) [102].

The involvement of a single genetic locus responsible for disease resistance or pathogenesis has been demonstrated for a few infectious diseases. Some good known examples in livestock are the dominant alleles responsible for receptors providing access to host cells for certain retroviruses in chicken [103], and entertoxigenic E. coli in swine [58].

Rasschaert, et al. found that the probability of an SR/SS genotype for MUCIN 4 increases significantly with increasing F4ab ETEC per 250 mm villi (P = 0.029), with the odds ratio for each unit increase of F4ab equal to 1.036 (95% CI [1.004-1.069]) [104]. For F4ac, the probability of an SR/SS genotype for MUC 4 increases significantly with increasing F4ac ETEC per 250 mm villi (P = 0.030), with the odds ratio for each unit increase of F4ac equal to 1.018 (95% CI [1.002–1.034]). This variance is due to the fact that some pigs do not have any expression (due to the lack of the gene) and that the expression of the F4 receptor genes varies when the gene is present [57]. Another factor, which could explain the individual variation is the age of the pigs. The villus length decreases with aging [104]. Various potential host receptors for F4 fimbriae have been described, including MUC4, MUC13, MUC20, ITGB5, and TFRC [4,10,109]. Grange and Mouricourt (1996) already demonstrated that F4ab can bind to a receptor of the transferrin family but there should also exist an additional receptor(s) for F4ac. As a consequence, not only MUC4 gene polymorphism but also the expression of these other receptor/s has or have to be included in the screening assay for F4ac/ab receptornegative pigs [105]. This has led to MUC4 gene polymorphisms in the intron 7 region being used as an important biomarker in the classification of the majority of piglets as resistant or susceptible to F4+ ETEC infections [110,111]. Consistently, Nguyen et al. confirmed that MUC4 polymorphisms and their candidate glycoprotein receptors are highly associated with the MUC4susceptible genotype [112]. In addition, Peng et al. and Zhang et al. revealed that alleles in MUCIN genes have a strong association with susceptibility to enterotoxigenic Escherichia coli F4ab/ac in the pig [106,107]. Similarly,

Ren et al. and Zhou et al. in our laboratory, both found that susceptibility/resistance toward ETEC F4ac is conferred by the MUC13 gene in pigs [4,10]. However, Schroyen et al. in their study on MUC13 and MUC20 reported that neither of these genes is related to ETEC F4ac susceptibility in piglets [113]. Goetstouwer et al. however, recently confirmed that MUC4 and MUC13 genotypes are not completely associated with F4ab/ac ETEC susceptibility and there are likely to be other receptors [114]. Further development concerning piglets’ susceptibility to ETEC F4 diarrhea, Fu et al. also in our laboratory, identified HEG1 and ITGB5 as two novel candidates and most promising genes that underlie F4ab/F4ac susceptibility to ETEC diarrhea considering their functions and positions in chromosome 13. This finding has provided novel evidence for understanding the genetic mechanism behind the risk of diarrhea in piglets.

Yan et al. demonstrated that susceptibility or resistance to ETEC F4 is correlated with whether or not brush borders of the pig intestinal epithelium bind to the bacteria as can be visualized by phase-contrast microscopy [108]. The SNPs and the candidate genes, which are confirmed to be associated with susceptibility or resistance are summarized in Table 2. Melkebeek et al. reported that porcine aminopeptidase N (APN) serves as a receptor protein for F4ac+ ETEC. APN, can promote intestinal epithelial cell endocytosis in F4Rs piglets and is involved in the induction of mucosal immunity [115].

Xia et al. recently, found that IPEC-J2 cells express APN and that F4 E. coli was able to adhere/ bind to IPEC-J2 cells in an APN dependent manner. Pre-incubation with APN polyclonal antiserum and anti-F4 fimbriae monoclonal antibody both reduced ETEC adherence to IPEC-J2 cells. Results from Y2H and pulldown assays also showed that FaeG binds directly to APN.

Xia et al. did not find an impact on APN-FaeG binding after treating samples with metaperiodate (NaIO4), according to them, this suggests that at least under their in vitro conditions, APN glycosylation does not play a significant role in FaeG binding [6].

However, Xia et al. concluded that the molecular details regarding APN-FaeG interactions and their roles in ETEC adherence await further experimentation [6].

On the other hand, Interferons (IFNs) as antiviral agents play vital roles in both adaptive and innate immune responses against viral infections [93]. Type III IFNs (IFN-λ) were first identified by Sheppard et al. in 2003 and found to be encoded by 1–3 functional genes in most species of mammals [94]. Among the

four interferons (IFNλ1, IFN-λ2, IFN-λ3, and IFN-λ4) found in humans [95,96] only two (IFN-λ1 and IFN-λ3) have been identified in pigs

[97]. Shen, et al. [98] showed that porcine interferon Type III (poIFN-λ3) has antiviral properties against PEDV and thus, may serve as an important bio-therapeutic tool for inhibiting PEDV in pigs.

Conclusions

Both pre-weaning and post-weaning diarrhea caused by Enterotoxigenic E. coli (ETEC) and porcine epidemic diarrhea virus in piglets remains major hurdles affecting economic growth in the piggery industries. Many studies have been carried out with the aim to have a better comprehension of the pathogenesis of the disease, which may lead to improved methods of prevention, disease prediction and treatment. The fact that the major virulence genes for ETEC are on plasmids means that various combinations of these genes are possible but there is a tendency for certain virulence genes to cluster together, suggesting that there are functional relationships that are not yet understood. ETEC of O-group 149 has been recognized as the dominant type of ETEC worldwide, but the basis for this dominance is not known. F4 and F18, the major fimbrial antigens present on the E. coli that cause PWD in pigs, have been identified as good targets for active and passive immunization and utilized for the selection of intestinal receptor-negative pigs.

Although diarrhea is a factor of huge economic loss in pig production, a few solutions or strategies have been put forward in attempts to mitigate the diarrhea problem. None of the attempted solutions or strategies to the problem of diarrhea in piglets has been consistently effective [8]. Newborn baby pigs may be protected by vaccination of their dams with pilus antigens that induce effective passive local immunity [116]. However, after weaning, loss of protective antibody in milk, transportation, mixing of pigs from several sources, fighting, and transition to a new diet all predispose pigs to diarrhea. Orally administered chicken egg-yolk antibodies against F4 and F18 fimbriae have failed to be effective when low doses were administered [117]. The most successful approach on a particular pig farm will probably involve combinations of molecular genetic tools, diet modification and other preventive measures.

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