MacConkey agar

Brilliant green agar

XLD medium

Triple sugar iron (TSI) agar

This is an indicator medium only and does not contain an inhibitor. A brief description of the medium and technique for inoculation is given in Chapter 2. It is prepared in tubes with a slant.

The general interpretation of the reactions is as follows:

The reactions are illustrated in Figure 17.16, while Figure 17.17 gives a summary of the differentiation of some of the enterobacteria by their reactions in TSI agar and lysine decarboxylase broth.

• Neonates obtaining insufficient passive immunity (antibodies) from colostrum. This might be due to either a quantitative or qualitative deficiency.

• Intensive husbandry practices lend themselves to rapid transmission of the pathogenic E. coli strains.

• Poor hygiene that allows a build-up of pathogenic strains in the environment of the young animal. A large dose of pathogenic E. coli may overcome colostral immunity.

• Young neonates, under one week of age, are particularly susceptible because:

• Recently weaned pigs are subject to stress factors such as altered surroundings, companions and diet. Heavy grain diets in particular can lead to a massive colonization of the anterior small intestine by enterotoxigenic strains of E. coli.

• Oedema disease occurs most commonly in young weanling pigs but the disease can occur in older pigs. The following factors are often present prior to the occurrence of oedema disease in pigs:

Enterotoxigenic E. coli

These strains cause the majority of cases of neonatal colibacillosis in calves, lambs and piglets. They do not appear to be an important cause of diarrhoea in other domestic animals, for reasons which are as yet unclear. Pathogenicity is correlated with the presence of adhesins and the production of enterotoxins. The toxins function by reducing absorption and increasing secretion without damaging the intestinal epithelium. The first step in the production of disease is the adherence of the ETEC to the intestinal epithelium. The structures by which the ETEC adhere are most commonly fimbriae and these are classified according to properties such as their amino acid composition and their ability to agglutinate red blood cells in the presence or absence of D-mannose. The fimbriae described in pigs and calves include F4 (K88), F5(K99), F6 (987P), F17, F18 and F41 (Nagy & Fekete 1999). ETEC strains are host-specific and usually cause disease in the first week of life only. Host specificity can be explained by the presence or absence of genes encoding for fimbrial receptors in the intestinal lining of the host. Age-related resistance may be due to the degree of expression of host receptors. It appears that some of the receptors are over-expressed as the age of the animal increases, thus leading to shedding of receptors into the intestinal lumen. These free receptors coat the ETEC strains in the intestinal contents, thus preventing their adherence to the intestinal lining (Dean et al. 1989). F18 receptors are not produced in newborn pigs but are increasingly expressed up to approximately four weeks of age, thus helping to explain why ETEC are a cause of diarrhoea in pigs up to this age.

Following adherence, ETEC produce protein enterotoxins. The toxins can be subdivided into two main groups: Large heat-labile toxins of approximately 88 kDa in size (LT) and smaller, heat-stable toxins (ST) containing 11–48 amino acids. Both toxin types have two subgroups. Porcine strains of ETEC produce mostly LT1 and Sta whereas strains of ETEC occurring in calves usually produce Sta. STb is associated with porcine strains of ETEC although it may be produced by some strains of ETEC isolated from calves, chickens and humans. LT has a similar mechanism of action to the heat-labile toxin produced by Vibrio cholerae. It consists of an A domain and five B subunits. The B subunits bind to the cell and part of the A domain then enters the endoplasmic reticulum of the cell and activates the adenylate cyclase system. The resulting increase in cAMP levels leads to increased fluid and electrolyte secretion and decreased absorption. STa acts by increasing guanylate cyclase activity leading to elevated levels of cGMP in the cell. Increased levels of cAMP or cGMP activate protein kinases which induce phosphorylation of the cystic fibrosis transmembrane regulator (CFTR). This in turn causes secretion of chloride and bicarbonate ions. Protein kinases also inhibit reabsorption of sodium ions (Dubreuil 2012). There is a resultant reduction in absorption of water and electrolytes at the villus tips and an elevated secretion of chloride and water in crypt cells. The mechanism of action of STb differs from that of LT and STa as it does not activate adelyate or guanylate cyclases but phosphorylates CFTR through a different mechanism. Interference with the enteric nervous system may also be important in the secretory diarrhoea induced by E. coli enterotoxins (Dubreuil 2012).

Enteropathogenic E.coli

These strains are included in the attaching and effacing E. coli (AEEC) because of the nature of the lesions they produce in the intestinal lining. Classical or typical EPEC strains were first described as a cause of diarrhoea in infants and these strains are strict human pathogens and possess a specific (EPEC) adherence factor plasmid (EAF) which is not found in animal isolates of EPEC. Atypical EPEC cause diarrhoea in piglets, lambs, calves and pups. The virulence factors of EPEC are encoded by a pathogenicity island known as the locus of enterocyte effacement (LEE). The genes on this island encode a number of components including the outer membrane protein intimin and its receptor, known as translocated intimin receptor (Tir). Tir is inserted into a host cell membrane where it functions as a receptor for intimin and thus allows close attachment of the E. coli cell to the host cell. A characteristic pedestal formation and effacement of microvilli then follow. In addition, other effectors produced by the bacterium cause increased levels of intracellular calcium, secretion of chloride ions, impairment of tight junctions and recruitment of neutrophils.

Shigatoxigenic E. coli

Oedema disease in pigs is often associated with E. coli O139 and O141 and these strains are usually haemolytic and produce Shiga toxin. These toxins are similar in activity to the Shiga toxin (cytotoxin) of Shigella species and inhibit protein synthesis in host cells following interaction with the 60S ribosomal subunit resulting in death of the cell. There are two types of Shiga toxins, Stx1 that is neutralized by antibody specific for Shiga toxin and Stx2 which contains a number of subtypes. Subtype Stx2e is associated with oedema disease in pigs whereas strains producing Stx1, Stx2, Stx2c and Stx2d are associated with haemorrhagic enteritis in humans. The toxin Stx2e is produced in the intestine but is absorbed and carried via the bloodstream to the target cells, usually endothelial cells of the small arteries. These oedema disease strains of E. coli are normally present in the large intestine of pigs, but they appear to multiply rapidly under conditions of stress, particularly a change of diet.

Enterohaemorrhagic E. coli

This group of E. coli strains is primarily of importance as a cause of food poisoning in humans, but may sometimes be associated with disease in animals including cases of haemorrhagic enteritis in calves. In addition to the LEE as described for EPEC, they contain other virulence factors including enterohaemolysin (Ehx) and phage-encoded Shiga toxin.

Septicaemic E. coli

Septicaemic (SEPEC) strains are responsible for septicaemia in their hosts. These strains may possess a wide range of virulence factors but few of these virulence factors are common to all strains (Mokady et al. 2005). It appears that each step of the disease process can be mediated by a number of alternative virulence factors and a particular invasive strain may have a unique combination of pathogenic attributes. Adherence to the intestinal lining is the first step in the invasion process. Adherence may be mediated by fimbrial adhesins, for example, F5 as in ETEC strains, by other fimbriae such as long polar fimbriae or by non-fimbrial adhesins. Septicaemic strains carry a plasmid encoding Colicin V (Col V plasmid). This plasmid encodes for type IV pili which have been shown to be important for adherence and invasion in Salmonella Typhi. In addition it encodes for serum resistance and the aerobactin iron uptake system, both important virulence factors for systemic survival of E. coli strains. Recently the presence of another iron uptake system has been demonstrated, similar to that found in Yersinia species, and dependent on the biosynthesis of the siderophore yersiniabactin. Different capsular types are present depending on strain and these are important for survival of the organisms following invasion. The immune response of the host leads to the death of some organisms and endotoxin is released, with resultant clinical signs of pyrexia, weakness, depression and tachycardia.

The diseases caused by E. coli in domestic animals are summarized in Table 17.4. Major virulence factors are given in Table 17.5.

Diagnosis of the septicaemic strains

The diagnosis, in this case, must be based on the isolation and identification of E. coli from normally sterile sites in the body such as bone marrow, joints, spleen or blood. Liver specimens should be avoided as there can be movement of enteric bacteria to the liver in the agonal stage of a disease.

Demonstration of the enterotoxigenic strains

The enterotoxigenic strains of E. coli are present in large numbers in the small intestine, and in this case it is insufficient to merely isolate and identify the E. coli. Demonstration of the significant fimbrial antigens (F4, F5, F6, F17, F18 and F41) or the enterotoxin itself is necessary. Alternatively, molecular detection of virulence genes can be employed.

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