Escherichia coli is a Gram-negative bacteria belonging to the family Enterobacteriaceae and is the causative agent of a wide range of diseases in pigs, including neonatal diarrhoea, post-weaning diarrhoea (PWD) and oedema disease (OD), which are important causes of death occurring worldwide in suckling and weaned pigs respectively (Zimmerman et al, 2019). OD is also known as ‘bowel oedema’ or ‘gut oedema’ because oedema of the submucosa of the stomach and the mesocolon is often a prominent feature of the disease. Shiga toxin encoding E.coli (STEC) strains result in release of Shiga toxin type 2e (Stx2e) which is the causative agent of OD in weaned piglets, with clinical signs usually becoming apparent around 2 weeks post weaning (Zimmerman et al, 2019). However, clinical problems and mortality can be observed during all nursery periods. Prevalence of STEC has been reported in various studies worldwide and has been shown to vary greatly, usually depending on genetics, farm management and antibiotic use in the post-weaning period (Tseng et al, 2014). Strategies to control OD include implementing a vaccination protocol against OD, which has been shown to be effective in reducing clinical cases and mortality caused by the disease.
The pathogen
E. coli is a bacterium that is commonly found in the lower intestine of animals. Non-pathogenic, intestinal E. coli (commensal E. coli) support the physiological intestinal balance of the host, whereas pathogenic E. coli with typical virulence factor gene profiles can cause severe outbreaks of different diseases.
OD is caused by Stx2e produced by a specific virotype of E. coli (STEC) (Figure 1). A virotype is determined by a particular combination of virulence genes. Important virulence factors encoded by STEC are the fimbrial adhesin F18 which is needed for attachment and colonisation of the gut and Shiga toxin (Stx2e) causing systemic disease. A different pathogenic E.coli is enterotoxigenic E. coli (ETEC). The ETEC carry the fimbriae F4 and F18, which again allow adhesion and colonisation of the gut, which results in production of enterotoxins including heat stable toxin a (STa), heat stable toxin b (STb) and heat labile toxin (LT) which cause post-weaning diarrhoea (Luppi, 2017). The pigs need to have specific porcine F18 receptors (F18R) on their enterocytes for the STEC to bind (Coddens et al, 2007).
While the E. coli encoding for Shiga toxin (Stx2e) occur world-wide, prevalence data particularly from herds without presence and history of ‘classical OD’ are rarely available. Nevertheless, a recently published longitudinal study in three US farms with healthy pigs observed a high prevalence (68%) of STEC in pigs raised for pork production. The usual detection rate of Stx2e in cases with clinical signs suspicious for OD is around 20%, up to 60% (Leneveu et al, 2019).
Shiga toxins (Stx) are a family of related toxins with two major groups, Stx1 and Stx2. The most common sources for Stx are the bacteria S. dysenteriae and the Shigatoxinogenic virotypes of E.coli (STEC). Both types of toxins have similar structures and modes of action (Tu et al, 2009). The toxins have two subunits: A and B (Figure 2). The A subunit is responsible for the enzymatic activity of the toxin including inhibition of protein synthesis. The B subunit is a pentamer that binds to specific glycolipids on the host cell, which allows the A subunit to enter the cell (Sato et al, 2013). Aft er a piglet is infected orally, STEC proliferates in the small intestine which results in the release of Stx2e and the development of systemic enterotoxaemia and clinical infection.
Pathogenesis
Pathogenesis of the OD can be divided into two phases: colonisation of the gut and initial enterotoxaemia, associated with changes in microbiota; followed by the systemic distribution of the Stx2e. The second pathogenic phase is responsible for the vascular damage, increased vascular permeability, fluid loss and tissue oedema on predilection sites (Tabaran and Tabaran, 2019).
The Stx2e passes through the intestinal wall into the blood and is transported by erythrocytes to the endothelial cells (Clugston et al, 1974). In the endothelial cells, the active site of the A subunit causes inhibition of protein synthesis, therefore leading to cell death. Receptors for Stx2e are present in vascular smooth muscle and their presence or absence is a major determinant in the tissue distribution of lesions.
F18 is one of the well-known adhesion factors which enables E. coli to attach to the intestinal mucosa of piglets. In the case of post-weaning diarrhoea pathogenic E.coli carrying F18 fimbriae colonise the small intestine by binding to the porcine specific F18R and cause postweaning diarrhoea or OD. Adherence of the bacteria to microvilli of epithelial cells in the small intestine is initiated by adhesins that are associated with F18 fimbriae. Colonisation depends on the specific binding between adhesive fimbriae and receptors on the enterocytes. Other ways of colonisation by STEC might be possible and STEC strains without F18 were isolated from clinical cases as well.
Stx2e encoding strains may possess either the fimbrial variant F18ab or F18ac. Susceptibility to colonisation and to infection with an E. coli F18 strain develops around weaning and is dependent on the presence of the porcine intestinal F18R genotype which is gradually increased aft er weaning. However, not all pigs have these epithelial cell receptors.
The expression of F18R is age-dependent and begins at around 3 weeks of age. Torrison (2011) described the main stages of the pig's life where E. coli encoding for Stx2e are detected with a peak in weeks seven to nine (Figure 3). Remarkably Stx2e positive strains can also be detected in later production stages, and it is generally the origin of serious clinical signs, increased mortality and impaired performance. Yet F18 is not detectable in every case of E. coli encoding for Stx2e (up to 20%). The relevance of these particular E. coli (without the F18 but with Stx2e) is nevertheless given, as they have been isolated as the only pathogen from cases with severe clinical signs.
Clinical signs
Usually, OD is considered mostly a weaned pig disease with signs occurring during the nursery period, usually within 2 weeks after weaning, although cases may be observed in later production stages as well. Stress at weaning is known to impair the immune system and lead to intestinal gut dysfunction in pigs (Rhouma et al, 2017). The disease may be sporadic and may affect only individual animals, but occasionally an entire batch of pigs is affected. It is important to emphasise that OD has a high mortality rate once clinical signs of infection are present, and usually affected piglets do not survive this stage of infection.
A subacute or acute outbreak oft en is recognised as sudden death without previous signs of sickness. OD is clinically characterised by swelling of the eyelids, fore head, unusual squeal or snoring sound, neurologic signs such as paresis, paralysis, ataxia, incoordination and recumbency and/or subcutaneous and sub-mucosal oedema in various tissues (Matise et al, 2000). As a result of the laryngeal oedema some pigs emit a peculiar squeal. Due to the oedema in the brain pigs show convulsions, ataxia and lateral recumbency with paddling of limbs (Figure 4). Few pigs survive the acute disease, but those that do remain runts (i.e. show stunted growth). The course of the disease can also be prolonged. For periods varying from days to several weeks aft er intestinal infection, growth stops and sick pigs oft en show unilateral nervous disturbances such as circling movements, twisting of the head, or atrophy of limb muscles with progressive weakness. In these cases sub-cutaneous oedema is rare (Zimmerman et al, 2019). Clinical signs reoccur in the same batch of pigs, especially after further change of feed, removal of zinc oxide (ZnO) or antimicrobials from the feed or another stress factor.
Subclinical OD can occur in clinically normal pigs which are colonised by Stx2e E. coli, which develop vascular lesions and may have a decreased growth rate (Zimmerman et al, 2019). These herds may show inhomogeneous groups of pigs in the different production stages. An experimental model of the subclinical form of OD was described by inoculation of 3-week-old piglets with 1010 colony-forming units of Stx2e positive E.coli isolated from pigs with clinical disease (Kausche et al, 1992). The negative effect of colonisation and presence of STEC strains on production results were described in the field on farms without obvious history of OD (Leneveu et al, 2019).
Diagnosis
Differential diagnoses of the disease in relation to central nervous system disorders include: pseudorabies; teschoviral encephalitis; Streptococcus suis or Haemophilus parasuis induced meningitis among others. Farms with suspected S. suis infections in nursery unit which are not responding well on treatment of choice, such as amoxicillin, should be evaluated for the the presence of STEC. It is important to note that with OD infection there will be no meningitis at histology. There are several non-infectious sources that may present similar clinical signs, such as water deprivation (salt poisoning), vitamin E/selenium or even stress due to handling. The disease should also be included as a differential diagnosis when sudden death is observed in the first weeks after the weaning (Zimmerman et al, 2019). For these reasons, diagnosis is an important tool to identify the main cause of disease and the conclusion should not be reached based only on clinical signs and farm history.
When completing farm sampling, the number of samples required depends on the expected prevalence of the disease and the total number of animals at each age on farm. Faecal samples and swabs can be taken for culture, but no more than five individual faecal samples should be pooled together. In order to confirm STEC circulation on farm usually six pools per five piglets from six different pens in the nursery is recommended. In deceased pigs, the collection of jejunum parts or swabs from the mucosa of the jejunum is highly recommended (Figure 5).
The first step with these samples is to perform the analysis of the bacterial culture. Once the E. coli strain is cultured, multiplex polymerase chain reaction (PCR) can be carried out for the detection of fimbrial and toxin genes. The advantage of the mulitplex PCR is to deliver a result on the spectrum of adhesion factors and toxins which enable the veterinarian to detect the E. coli strains involved and to make the correct diagnosis. It is necessary to provide the laboratory with a complete clinical history, including the clinical signs observed in the affected animals and the presumed aetiological cause.
During the post-mortem examination, the typical diffuse, vasculogenic oedema, occasionally accompanied with petechia, can be present in most tissues. Most often, the oedema affects the digestive system (gastric lamina propria and submucosa, spiral colon wall, meso-colon, small intestine mesentery and mesenteric lymph nodes and gallbladder), skin and subcutis (palpebrae, frontal skin, submandibular, ventral abdomen) and adjacent lymph nodes, thoraco-abdominal and pericardial serosa (Tabaran and Taberan, 2019).
Histopathological study of the samples, particularly the brain and distal jejunum, would help to clarify the diagnosis if the lesions of the general pathology are unspecific, and also to assess the relevance of the results obtained by culture and PCR. By microscopic examination, lesions of oedema, haemorrhage and vasculitis are noted. Microscopic lesions are associated with vascular injury and include vessel necrosis, perivascular oedema and haemorrhage, and superficial colonic and caeca erosions. The vascular lesions are observed in the cerebellar folia, submucosa and mucosa of the stomach, caecum, colon, and sporadically in the retina (MacLeod et al, 1991).
Control and protection
Control and prevention of OD can be difficult, as actually, few commercial vaccines or effective treatments are available. Nutritional changes can be made which can help to reduce colonisation with E. coli in pigs after weaning. For example, supplementation of piglets' diet with ZnO can reduce post-weaning diseases through reducing E. coli adhesion and intestinal permeability (Roselli et al, 2003). The use of high levels (pharmacological level) of ZnO post-weaning will be banned in countries in EU mid 2022, thus other strategies will be needed. Feeding of high energy and protein content that is administered after weaning causes an increase in the pH of the intestine that favours the multiplication of E. coli. Therefore, limiting the amount of feed or ad libitum feeding of high fibre and low protein diet can modulate fimbrial receptors (Kelly et al, 1994), which can reduce colonisation by E.coli after weaning. However, these strategies usually limit growth potential of modern pig genetics. Antibiotics have historically been used to control outbreaks, however due to the rapid course of the disease, treatment is often too late for piglets with clinical signs. The most effective antibiotics and classes of antibiotics frequently used post-weaning, such as colistin or fluorochinolones, belong according to new European classification to category B (‘restrict’). Antibiotics in this category are critically important in human medicine and their use in animals should be restricted to mitigate the risk to public health. The best results and optimal OD prevention and reduction of mortality have been achieved with vaccination, with vaccines containing the modified, and therefore non-toxic, Shiga toxin (Casanova et al, 2018).
Vaccination
The Shiga toxin vaccination reduces the mortality and clinical signs of OD into the finishing period. The single-dose of vaccination can be used from 4 days of age. Three weeks after the vaccination animals are protected by neutralising antibodies, which persist until at least the 15th week of life and protect animals in the most vulnerable period of life.
Nothing is as natural as the pig's own immunity, but the in-herent immunity against Shiga toxin can be actively boosted by vaccination. These antibodies neutralise the Shiga toxin and therefore prevent the development of acute or chronic clinical signs. However, the presence and colonisation of animals by Shiga toxin producing E. coli in the herd is not reduced by the vaccine because it does not act against the bacterium itself. While the vaccination itself almost eliminates the rate of losses and runts resulting from OD, additionally the farmer can dramatically reduce the use of antimicrobials or ZnO for controlling STEC in pigs. The Shiga toxin vaccination helps to minimise the risk of developing resistant bacteria and thus actively contributes to consumer protection. Compliance and efficiency of vaccination is easy to control through use of an in-house serum neutralisation test. The result of such a test corresponds directly to protection of piglets after vaccination.
In Europe, a vaccine based on a recombinant and genetically modified toxoid has been developed, which consists of a subunit in which the antigen is the genetically modified Stx2e recombinant toxin. The vaccine can be given to piglets from 4 days of age. In a field study, Ecoporc Shiga has been shown to be effective in preventing clinical presentation of the disease and significantly reducing of mortality due to OD. The farm had a history of reoccurring outbreaks of OD and an average mortality rate of 7.7%, and implementation of the vaccine meant there were no more losses due to OD and the farm was able to profit from production again. Furthermore, the farm was able to completely stop the use of colistin sulfate and reduce the total use of antimicrobials, helping towards achieving the reduction in use of antimicrobials in the livestock industries. Because of this, the farm was able to return to the green category of the antimicrobial surveillance programme (Fricke et al, 2015).
Another study was completed between December 2011 and April 2013 in Germany, where 179 farms were vaccinated against OD using Ecoporc Shiga (Lillie-Jaschniski et al, 2016). Average mortality reduced from 8.5 to 2.2% and the use of antibiotics reduced from 4.0 to 1.6 days before and after vaccination respectively.
Conclusion
OD caused by STEC strains and the Stx2e toxins they produce can have large impacts on farm due to decreased growth and increased mortality in weaner pigs. Effective vaccination based on toxoid vaccine is a proven effective tool for control of OD which significantly reduces consumption of critically important antibiotics in the most sensitive period for piglets post weaning.
KEY POINTS
- Shiga toxin encoding Escherichia coli (STEC) strains result in release of Shiga toxin type 2e (Stx2e) which is the causative agent of oedema disease (OD).
- Susceptibility to colonisation and to infection with an E. coli F18 strain develops around weaning and is dependent on the presence of the porcine intestinal F18R genotype.
- Clinical signs of OD include swelling of the eyelids, forehead, unusual squeal or snoring sound, neurologic signs and/or subcutaneous and submucosal oedema in various tissues.
- Faecal samples and swabs can be taken for culture and histopathology can be carried out to confirm diagnosis.
- Vaccination has been shown to be effective in preventing clinical presentation of the disease and significantly reducing mortality due to OD.