13. The Ribosome of Escherichia coli.

(a) The ribosomal proteins.

Most studies on bacterial ribosomes have been carried out on the E. coli ribosomes. They are comparatively large particles consisting of proteins and RNA. About 38% of the ribosome is protein and 62% is RNA. The ribosomal proteins are usually designated as r-proteins and the ribosomal RNA as rRNA. The ribosomes consist of two subunits the smaller or 30S particle and the lager or 50S particle. The r-proteins associated with the 30S particle are designated with the prefix S and a number from S1 to S21, while the those associated with the 50S particle are L1 to L34.

This original desgnation has been modified when it was realized that L8 is an aggregate of L7/L12 and L10. Also L7 and L12 are identical as are L26 and S20. The current view is that all the r-proteins and rRNA molecules are present in the ribosomes as single units, i.e. with a stoichiometry of 1:1 apart from L7/L12 of which there are probably four copies per ribosome. It also appears that all the ribosomes have the same composition of 52 r-proteins and that there are no "specialized" ribosomes. The amino acid sequences of all the proteins have been determined and their molecular weights established. They vary from a molecular weight of about 5000 with 46 amino acids to a molecular weight of 61,000 and 557 amino acids, with an average molecular weight of about 15,000.

There is little sequence homology between the r-proteins, suggesting their independant origins. It has been suggested that the ribosome originated predominantly from its rRNA components, with a stepwise addition of the r-proteins. Most of the r-proteins appear to be globular in shape, apart from S1, S4 and S8, which are elongated.

(b) The ribosomal RNA.

The fact that nearly two thirds of the ribosomal structure is RNA and the suggestion that RNA was the original component of the ribosome onto which the proteins were built strongly indicates that it has more than just a structural role to play in the functioning of the ribosome.

There are three rRNA molecules associated with ribosomes. There is one molecule, a 16S RNA strand, associated with the 30S subunit, and two molecules of 5S and 23S associated with the 5OS subunit.

The 5S rRNA strand is composed of 120 nucleotides, none of which have been modified after transcription. In its secondary structure it appears as two predominantly double helical loops attached to a double helical stem, all three being of about similar length.

The larger 16S rRNA consists of 1542 nucleotides. Ten of these, at positions 527, 966, 967, 1207, 1402, 1407, 1498, 1516, 1517 and 1518 are methylated. These methylations have been found to occur at the phylogenetically most conserved regions of the 16S RNA and therefore are considered to have an important funcional role. The secondary structure shows a complex arrangement of double helical loops and stems.

The largest of the three is the 23S rRNA, which consists of 2902 nucleotides. It contains many post-transcriptionally modified nucleotides, including pseudouridines and methylated bases. Again the secondary structure indicates a highly complex arrangement of stems and loops.

In all these rRNA species there are indications for the existence of complex tertiary and quaternary interactions, which are likely to play a major role in the functioning of the ribosomes. It should never be forgotten, that these are the structures on which all proteins are made by the E. coli and have to be accurately transcribed from the mRNA.

(c) Assembly of the Ribosomes.

As can be inferred from the structure of the individual components of the ribosomes the internal structure of the ribosomes is highly complex. However, it has been demonstrated, that intact and functionally active ribosomes can be formed in the laboratory by simply putting all the components together. This was first demonstrated with by incubating 16S rRNA with a mixture of the S-r-proteins under conditions of high ionic strength. Using a two step method the reconstitution of the 50S subunits of E. coli ribosomes was similarly achieved.

Using the simple logic that a protein, which does not bind well to the rRNA but does bind well to the rRNA after another protein has been bound, to indicate that the former protein would follow the latter protein in the assembly process, a systematic sequence of the ribosomal subunit assemby has been established. These assembly stages have been mapped. Further studies have shown that these sequences actually follow the temporal sequence very closely.

Although large rRNA molecules and many proteins are involved, the kinetics of assembly follow first order kinetics, indicating that the rate-limiting step is the reaction involving one molecule. An essential feature of the essembly of the ribosomes is the cooperative binding of the r-proteins, with the binding of some being dependent on either prior or simultaneous binding of others. Studies also indicate this strong cooperative action in ribosomal assembly within the E. coli cell.

The assembly of 30S ribosomal particles can occur in the absence of 50S ribosomal particles and vice versa. Within the cell there is an interaction between the two assemblies, with an excess of r-proteins of one subunit inhibiting the assembly of the other.

(d) The Structure of Escherichia coli Ribosomes.

There have been extensive studies using electron miscroscopy and other means of visualising such particles, to elucidate the three diemnsional structure of the E. coli ribosome. While there are some discrepencies between the models obtained by the various methods, certain specific aspects of the structure are now established.

The 30S particle has been shown to consist of a "body" comprising about two thirds of the particle and a "head", comprising the remaining third. In some studies a "platform"-like projection can be seen, extending from the body near the body-head junction. There is "cleft" between the head and this platform. The largest length is about 24-25 nm.

The 50S particle, which is more sperical in shape, nevertheless has three obvious projections pointing outwards. On one side of a "central protuberance" is the "L7/L12 stalk" and on the other the "L1 ridge". Again the largest length is about 24-25 nm.

It appears that in the combined 70S particle the platform side of the 30S particle faces the concave side of the 50S particle. The gap between the central protuberance and the L1 ridge seems to be, where the head of the 30S particle fits into.

By means of some biochemical studies the positions of some of the proteins and RNA relative to each other have been mapped. Suffice it to say that the ribosome is a very complex structure.

14. Protein Synthesis in Escherichia coli.

(a) The First Stage.

The first stage is the so-called translation of the amino acid to the amino acid-tRNA complex. This involves two steps. In the first step the enzyme aminoacyl-tRNA synthetase (RSi) activates the specific amino acid (aai) in the presence of ATP to form an amino acid-adenylate (aai-AMP), releasing inorganic pyrophosphate (PPi). This activated aai-AMP while still compexed to the RSi now combines with the 3'-terminal adenosine of the appropriate tRNA (tRNAi) to form the specific aminoacylated tRNA (aai-tRNAi). The net reaction is thus that the aai reacts with ATP and RSi to form the aai-tRNAi releasing the RSi and splitting ATP into AMP and PPi.

It has been estimated that around 15% of the total RNA of an E. coli cell is made up of tRNA. This figure can be extrapolated to about 10-15 tRNA molecules per ribosome or about 400,000 per cell. The structure of the tRNA's varies from 74 to 94 nucleotides. They comprise the usual nucleotides; adenine, guanine, cytosine and uracil, which are typically found in RNA, but about 10% of the nucleotides are modified after the transcription of the nascent tRNA. About 50 different modified nucleotides have been identified among the tRNA species.

It was realised as soon as the nucleotide sequences of some of the tRNA's had been determined, that they are likely to be folded into a number of helical hairpin and loop conformations. In two dimensions, they are often shown in structures closley resembling a cloverleaf. The three dimensional structure shows an 'L' shaped configuration with the loop containing the exposed anticodon triplet at one end and the 3' end, which accepts the amino acid at the other end.

The aminoacyl-tRNA synthetases have a great deal of similarity in both secondary and tertiary structure. Though these do not apply to the primary amino acid sequences. It has been shown that each aminoacyl-tRNA synthetase recognizes and then aminoacylates the members of its cognate isoreceptor tRNA family. It also discriminates against all other tRNA's. It has been shown that changing a single nucleotide in the tRNA molecule can change its recognition from one aminoacyl-tRNA synthetase to another. While some aminoacyl-tRNA synthetases react with the anticodon region in determining their specificity this is not the case for all.

(b) The Second Stage.

The second stage of protein synthesis is considered the Initiation stage. This involves a number of reactions, which must occur between the formation of the aminoacyl-tRNA's and the formation of the first peptide bond, linking two amino acids correctly together. The ribosomes, which play the essential role in this process first dissociate into the 30S and 50S subunits.

Prior to initiation, the 30S subunit forms a preinitiation complex with the mRNA and a unique tRNA molecule. This unique tRNA is N-formyl-methionyl (fMet)-tRNAMetf (fMet-tRNA). Subsequently the 50S subunit joins to form the 70S initiation complex. Three proteins, known as the initiation factors IF1, IF2 and IF3 as well as GTP, which is hydrolysed to GDP and inorganic phosphate are required for initiation. Thus, the correct stretch of mRNA is selected so that the correct message is translated, also the mRNA is translated in the correct sequence so that the correct reading frame is used. With the triplet code, it has to be realized that the mRNA must be read such that the first letter of the triplet is recognized as such, otherwise if the second or third letters are thought to be the first letter then nonsense will be produced and this is considered as using the wrong reading frame.

The initiation of all bacterial protein synthesis starts with an fMet at the N terminus. Posttranscriptionally the formyl group and sometimes the methionyl residue itself is removed. There are two tRNA's which recognize methionine. They are tRNAMetf , which is specifically involved in the initiation of protein synthesis and the tRNAMetm , which specifically provides the methionine when required as part of a protein. Both these tRNA's recognize the unique AUG code, which is specific for methionine, having the anticodon CAU. While both are aminoacylated by the same methionyl aminoacyl tRNA synthetase, only the Met-tRNAMetf is formolised to fMet-tRNAMetf .

The formylation of the fMet-tRNAMetf is required for the efficient initiation of protein synthesis in E. coli. In bacteria, like E. coli, the mRNA sequence being translated is often polycistronic. This means that a number of separate proteins are being synthesized in sequence and therefore, there will be a number of stop and start signals. Additionally the translation of the mRNA is "coupled" to the transcription of the mRNA from the DNA. The ribosome initiates translation shortly after the first initiation site on the mRNA emerges from the RNA polymerase and well before the transcription of the complete mRNA message is released.

The most frequently used initiation triplet codon is AUG, and GUG is used for about one in ten initiations. Of the others UUG is used even less frequently and AUU very rarely. In front of these initiation codons is a nontranslated region. The three initiation codons AUG, GUG and UUG all have been shown to promote the fMet-tRNA binding to the 70S ribosomes. They will also all act as reinitiation sites, if they follow nonsense codons. Upstream of the ribosome binding site is a purine rich region containing the sequence GGAGG, which probably base-pairs with the 3' terminus of the 16S rRNA of the 30S ribosomal subunit. There are other bases in the vicinity of the initiating site, which control strength of binding and intiation. Throughout these processes the extensisive set of fine-tuning involved is becoming ever more clear.

Of the proteins involved in initiation, IF1 and IF3 are small basic proteins. There are two different forms of IF2, known as IF2a and IF2b, which are larger acid proteins. IF1 promotes the functions of IF2 and IF3, IF2a and IF2b bind the fMet-tRNA and have the GTPase activity and IF3 causes the 70S ribosome dissociation and mRNA binding. The 70S ribosome is in equilibrium with its components. The initiation procedure can thus be summarised as follows below.

The 70S ribosome dissociates in the presence of IF1, IF2, IF3 and GTP and these four bind to the 30S ribosomal subunit. The mRNA and the fMet-tRNA bind to this complex, such that the anticodon of the tRNA couples with the initiation codon on the mRNA, with the release of IF3. The 50S ribosomal subunit now recombines with the 30S ribosomal subunit, expelling the IF1 molecule and causing the hydrolysis of the GTP to GDP and inorganic phosphate and release of IF2. The 70S ribosome has the mRNA in position with the fMet-tRNA coupled to the initiation codon ready the bind the next amino acid according to the sequence of the mRNA and the ribosome ready to accept this next aminoacyl-tRNA.

(c) The Third Stage.

The addition of the second as of all subsequent aminoacyl-tRNA's follows the same sequence and is known as the Elongation cycle. This cycle has for convenience been subdivided into three stages, which are the binding of the aminoacyl-tRNA, the peptide bond formation and the tranlocation. All these reactions should be seen as occurring on the surface of the ribosome and involve two binding sites for tRNA. These are the P-site, where the peptidyl-tRNA binds and the A-site, where the aminoacyl-tRNA binds.

Apart from the ribosome, the peptidyl-tRNA (or in the case of the second aminoacid to be added, the bound fMet-tRNA) and the incoming aminoacyl-tRNA there are a number of other factors involved. These are the Elongation Factors EF-Tu, EF-Ts and EF-G as well as GTP.

The protein Elongation Factor EF-Tu, which comprises about 5% of the total soluble protein of an E. coli is the most abundant E. coli protein. In a rapidly growing E. coli cell, the ratio of EF-Tu to ribosome is about 7:1, while the EF-Tu:tRNA ratio is about 1:1. The EF-Tu molecule has an acetyl group on the N-termal serine residue and in about 60% of the molecules there is a methyl group attached to the lysine in position 56. In the absence of GDP the purified EF-Tu molecule is very unstable. It has 393 amino acids and is about 43 kDa.

The protein Elongation Factor EF-Ts, with only 282 amino acids is slightly smaller being about 30 kDa. It has been purified as a EF-Tu:EF-Ts complex from which it can be separated by the addition of GDP.

The protein Elongation Factor EF-G is the largest of the protein Elongation Factors, having 703 amino acids and being about 80 kDa. It is an acidic protein. Within an E. coli cell there is about one EF-G protein per ribosome and it comprises about 0.5% of the total soluble protein of a rapidly growing E. coli cell. It binds both GTP or GDP.

The first part of the elongation cycle consists in the formation of a ternary complex of the aminoacyl-tRNA with GTP and EF-Tu. It is this intermediate, which binds to the ribosome. In its formation the EF-Tu first forms a binary complex with GTP, which subsequently forms the ternary complex with the aminoacyl-tRNA.

This ternary complex now reversibly binds to the A-site of the ribosome. The specificity of this binding is determined by the codon-anticodon interaction. It is the formation of hydrogen bonds between the the three anticodon bases of the tRNA and and the three codon bases of the mRNA, which determines the specificity of the binding and ultimately the specificity of the translation of the mRNA message into the amino acid sequence of the protein being formed.

With ternary complex in place, peptide bond formation can now occur. The peptidyl-tRNA, which is in the P-site is an ester bond, which donates its peptidyl group to the amino moiety of the aminoacyl-tRNA in the A-site to form instead a peptide bond. The energy for this reaction is provided by the coupling of the reaction with the hydrolysis of GTP to GDP and inorganic phosphate, catalysed by EF-Tu, acting as a GTPase. The newly formed EF-Tu:GTP complex is now released from the A site. The reaction is mediated by an enzyme peptidyltransferase, which is an integral part of the 50S subunit of the 70S ribosome. The newly enlarged peptidyl-tRNA molecule is now in the A-site and for the next amino acid to be added it must vacate this site and move to the P-site. This process is known as translocation.

Elongation Factor EF-G is the component involved in the process of translocation. It, too, is associated with GTP hydrolysis. Firstly EF-G forms a binary complex with GTP. This binds to ribosomes, in which there is a stripped tRNA in the P-site and a peptidyl-tRNA in the A-site. As a result of the binding of this binary complex the stripped tRNA is released from the P-site. Then the peptidyl-tRNA is moved to the P-site, leaving an open A-site ready to receive a new ternary complex of EF-Tu:GTP:Aminoacyl-tRNA. At the same time the GTP is hydrolysed to GDP and inorganic phosphate and the EF-G:GDP complex is ejected from the ribosome. The movement of the mRNA so that the new codon bases are exposed in the A-site probably occurs with the movement of the peptidyl-tRNA from the A-site to the P-site.

The ribosome is now ready to receive a new ternary complex. Also in the meantime, the ejected EF-Tu-GDP complex in association with EF-Ts ejects the GDP and forms a new complex with GTP, ready to form the ternary complex with another aminoacyl-tRNA. Similarly the EF-G splits from the GDP and forms a complex with GTP. Thus amino acid after amino acid is added to the growing peptide chain.

When the ribosome reaches one of three termination codons, UAA, UAG or UGA then termination occurs. Again there are proteins involved in the termination and release of the peptide, which are known as release factors. Release Factor 1 (RF-1) with 323 amino acids is about 36 kDa. It reconizes the termination codons UAA and UAG, while RF-2 with 339 amino acids is about 38 kDa and recognizes the termination codons UAA and UGA. The protein RF-3, which has GTPase activity and stimulates RF-1 and/or RF-2 is about 46 kDa and the protein RRF which causes the ejection of the tRNA is about 23 kDa. There are about 500 to 700 release factor molecules per E. coli cell, being less than 10% of the initiation and elongation factors.

Termination commences, when one of the termination codons reaches the A-site, with the just completed peptide chain still attached to the last tRNA in the P-site of the ribosome. The appropriate release factor, RF-1 or RF-2, now attaches to the termination codon. The termination factor RF-3 enhances this binding although itself not recognizing the termination codons. This binding of the release factors promotes the hydrolysis of the peptidyl-tRNA by the peptidyl transferase. The peptide is now released from the 70S ribosome, which still holds the mRNA, the stripped last tRNA in the P-site and the release factor in the A-site.

As a result of the hydrolysis of the of GTP to GDT and inorganic phosphate by RF-3, the GDP formed causes the dissociation of the RF-1 or RF-2 from the termination codon. It is the action of the RRF which stimulates the release of mRNA and tRNA from the ribosome. Thus, the ribosome is ready to start again.

15. Human Intestinal Diseases caused by Escherichia coli.

General considerations.

With the intestinal tract being the main habitat of E. coli it is perhaps not surprising, that these organisms have also evolved some virulence characteristics which permit them to become pathogens within this environment. The initial pressure to develop or acquire these characterists may have been associated with the need to compete with either other E. coli, other bacteria or both in this highly competitive environment. The type of human diseases caused by E. coli have classified into the two groups specific and non-specific infections.

The main characteristic of specific infections is that colonization of a specific surface tissue is an essential prerequisite to the development of the infection. The types of disease which then develop are a direct result of specific interaction of the E. coli and their virulence factors at those sites.

In non-specific infections there is no direct involvement of the E. coli on a certain site, but they may be due to some contamination such as of wounds or due to some underlieing factors, which have compromised the host such alcoholism or immune defficiency.

There are a number of different mechanisms of pathogenesis, which E. coli can utilise within the human intestinal tract. For most of these some form of colonisation of the mucosal surface of the intestinal tract is an essential prerequisite. While in the first half of this century, there were a number of suspicions, that E. coli might have a pathogenic potential, it was always difficult to prove. The problem basically was the difficulty of distiguishing the apparently pathogenic E. coli from the commensal E. coli. There was also the intellectual problem of accepting that an organism as widespread in the human and animal intestinal tract and generally non-pathogenic, could also be pathogenic. In fact the main primary differentiation of the then recognized intestinal pathogens like Salmonella and Shigella from the commensal E. coli was that while the former did not generally ferment lactose, the lattter did. This created a very useful primary isolation medium for the identification of these pathogens, but specifically excluded even consideration of these lactose-fermenting E. coli as pathogens.

It really was the development of the serotyping scheme for E. coli, which first created the ability to characterise different clones of E. coli. It was an outbreak in the 1940's in Scotland of infantile diarrhoea, that led to the first demonstration that certain types of E. coli can cause human disease. These E. coli came to be known as the Enteropathogenic E. coli (EPEC).

16. Enteropathogenic Escherichia coli (EPEC).

(a) Introduction.

Infantile gastroenteritis or summer diarrhoea has been known for many years as a major, cause of morbidity and mortality among young children, particularly those under the age of two years. While very uncommon in the developed world at present, it is still a major problem in the developing world. It was realized that certain serotypes of E. coli are predominantly associated with this condition but for a long time no mechanism for the way E. coli can cause this disease was discovered. If Enteropathogenic E. coli (EPEC) were suspected as being the cause of a particular outbreak they were sent to a specialised laboratory for serotyping and if they belonged to one of the few serotypes, which had been found to be associated with this condition they were considered as EPEC.

The difficulty with this identification of EPEC was that it did not take into consideration that not necessarily all members of a so-called EPEC serogroup were EPEC. This was certainly the case. Also not all EPEC serogroups had necessarily been described and regularly new serogroups were reported from around the world. Most importantly this way of describing EPEC gave no information at all about the mechanisms of action of EPEC.

(b) Early Studies on Virulence of EPEC.

The possiblity that certain serogroups of E. coli may be EPEC had already been established, when an experiment was performed in the 1950's in which approximately 108 organisms of a possible EPEC strain of E. coli which had been isolated from children with diarrhoea, aged between one and three months, were fed to a two month old infant with multiple congenital defects. This resulted within 24 hours in diarrhoea, weight loss and extensive colonization of the gastrointestinal tract and other areas. Subsequent treatment of the infant with the antibiotic terramycin resulted in clinical improvement and eliminated the strain from the infant. More recently it was established that EPEC serotype strains isolated from ill infants can induce diarrhoea in adult human volunteers.

In the 1950's and 1960's the epidemic proportion of EPEC outbreaks in the developed world reached the point that a number of countries were forced to employ special measures to deal with the situation. Currently in the developing world similar serotypes have been found associated with infantile diarrhoea, however techniques have generally not been available there for the tests to be performed. In those few instances, where they have been carried out similar serotypes have been identified.

As serotyping was the only way of distinguishing EPEC from other E. coli it required laboratories to have batteries of antisera to test for EPEC. Due to the cross reactions of many E. coli serotypes this was very labour intensive and sera had to be carefully tested and absorbed. It was hoped that significant advance would be the use of fluorescein labelled antiserum pools to identify EPEC, but when fluorescent labelled sera were used to identify EPEC in swine it was found that 80% of specimens taken from animals on the farm as well as from carcasses after slaughter which were positive using the fluorescent antibody technique did not yield EPEC by culture. This confirmed the difficulty of relying solely on these rapid methods. It is also a lesson for today when in some cases inadequate techniques are still used to identify human pathogenic E. coli from animals.

The EPEC have traditionally been identified on the basis of their belonging to certain serotypes, which have been classed as the EPEC serotypes. The definition of a strain belonging to an EPEC serotype is based on it having both the 'O' and 'H' antigen that has been established over the years as associated with these EPEC types. However, often laboratories only define EPEC on the basis their reaction in the 'O' sera. More recently an EPEC-adherence factor (EAF) has been identified, which is the adhesin responsible for the localized adherence (LA) pattern to Hela and HEp-2 cells. These are human derived cells, that can be grown easily in tissue culture are are often used to study the interactions of microorganisms and human cells. This EAF appears to be strongly associated with the classical EPEC serotypes.

(c) Recent Studies on Virulence of EPEC.

Recently strains of E.coli were isolated from the stools of 500 infants with diarrhoea less than one year of age and an equal number of age-matched children with no gastrointestinal symptoms for at least 30 days prior to the specimen being taken. The isolates were confirmed as being E.coli biochemically and were examined by means of antisera as to whether they belonged to the EPEC or other currently accepted diarrhoeagenic serogroups. The 'H' antigens of all strains belonging to the EPEC 'O' groups were identified and they were tested to determine if they were EAF+.

This study revealed that EAF+E.coli were strongly associated with diarrhoeal disease and most of these EAF+E.coli were EPEC serotypes. In addition it revealed that EAF+E.coli are mainly but not exclusively members of EPEC serogroups and most frequently these are associated with diarrhoeal disease and when 'H' typing was performed are members of the classical EPEC serotypes. However, neither EAF determination nor EPEC serogrouping alone can substitute for careful and detailed EPEC serotyping. Detailed studies on strains of E.coli from diarrhoeal disease which may be EPEC should be done and these should include full 'OH' serotyping and EAF determination.

Currently in the developed world EPEC do not appear to play the important role as a pathogen of young children, which they did in the past, however in the developing world they are still very important. In a survey of children with diarrhoea in Iran, EPEC were the most common cause of diarrhoea among children accounting for nearly 31% of cases. The serotypes. A recent study in Nairobi, Kenya has demonstrated the importance of EPEC belonging to the serogroup O111. Unfortunately their H antigen did not belong to one of the accepted H antigens. In this study in a hospital in Nairobi, Kenya of an outbreak of diarrhoea in November-December 1985 in nursery ward for preterm babies aged between 4 and 64 days, cultures were obtained from 30 babies. Standard microbiological procedures were employed to identify the usual enteropathogens. In addition plasmid DNA was isolated and the strains grouped according to their plasmid profile analysis. DNA probes against the EAF gene as well as against the enterotoxins of Enterotoxigenic E. coli (ETEC, see below) were used to identify strains of E.coli producing these virulence factors. The isolates were also tested for their ability to produce Verocell cytotoxin, for their adherence to HeLa cells and they were serotyped.

Over two thirds of the lactose fermenting colonies isolated from the 30 babies were found to be E.coli and the remainder were Klebsiella pneumoniae. None were found to produce the accepted enterotoxins. The plasmid profile found amongst the largest number of strains was isolated from 13 infants. All those strains were positive for the EAF probe, HeLa cell adherence and belonged to serogroup O111.Hnt. Four of the 13 infants, who harboured this type died. The EAF gene was found on a 65-megadalton plasmid This study demonstrates the importance of being able to identify such EPEC, however all current tests for EPEC are either time-consuming, expensive and certainly not fully reliable.

In 1966 an observation was made that chloroform-killed suspensions of EPEC could cause a similar production of fluid in a rabbit ligated gut-loop as living cultures of EPEC was the first demonstration that a 'toxic' agent or agents may be involved in infantile gastroenteritis. This 'toxin' is not the same as any discussed below and requires further investigation.

In a recently described test use is made of two phenomena. EPEC are known to adhere to the intestinal mucosa to produce the characteristic 'attaching and effacing' lesion in the surface of the membrane, whose microvillus structure essential to aid absorption of material from the intestine. It is the destruction of this site, which is characteristic of the 'attaching and effacing' lesion, which causes the fluid loss and hence the diarrhoea. Fluorescein-labelled phallotoxin (FITC-phalloidin) specifically binds to filamentous actin. Actin is a protein invoved in cellular movement. It is the actin in muscles, which causes them to move. A combination of these facts uses the FITC-phalloidin to identify the dense condensations of microfilaments produced when EPEC adhere to the HEp-2 tissue culture cells. Ten known EPEC strains and ten non-EPEC strains were tested and it was found that only the EPEC gave the characteristic intense spots of fluorescence, which corresponded with each adherent bacterium. Even EPEC previously described as non-adherent to HEp-2 cells were weekly adherent in this test. This test has proved in subsequent studies to be the specific test for EPEC that has so far been lacking.

(d) EPEC as Animal Pathogens.

(I) Pigs

ETEC and EHEC appear to be the main E. coli pathogens of pigs rather than EPEC.

(II) Cattle

ETEC and EHEC appear to be the main E. coli pathogens of calves rather than EPEC.

(III) Sheep

ETEC and EHEC appear to be the main E. coli pathogens of lambs rather than EPEC.

(IV) Goats

There are no reports of EPEC affecting goats.

(V) Horses

Foals are often prone to diarrhoea, but there are very few reports of EPEC being involved. A report showed that EPEC of human origin and belonging to serotype O111:H- affected to equine intestine.

(VI) Poultry

E. coli are associated with a number of diseases of poultry, but there do no appear to be any reports of EPEC-related conditions in poultry.

(VII) Dogs

There are very few reports of EPEC affecting dogs. An E. coli serotype O49:H10 found in a puppy may have been an EPEC.

(VIII) Cats

There are very few reports of EPEC affecting cats.

(IX) Rabbits

EPEC appear to be the major cause of diarrhoea among rabbits. Stock losses as high as 40% have been reported as a result of an EPEC outbreak. EPEC have been reported as the second most significant pathogen in commercial rabbit farming. The EPEC associated with rabbit diarhoea are often described as rabbit diarrhoeal E. coli (RDEC) and this term is frequently used in the literature.

Certain sero- biotypes appear to be more pathogenic for suckling rabbits, while others predominantly for weaned rabbits. It has been suggested that these different types represent different clones with varying virulence factors. There is also some geographical disparity among the different serotypes.

Of the adherence mechanisms are involved, it appears that special fimbriae play an important role. These have been termed AF/R1 (adherence fimbriae, rabbit 1), but as they have not been shown to be present in all RDEC there must be other factors. There is even some doubt, whether in those strains of E. coli, which do carry them, they are the main adherence factors. More recent studies suggest that an outer membrane protein similar but not identical to the eae protein of human EPEC may be involved. A majority of RDEC give a positive FITC-phalloidin test strongly indicating an important role for this attachment mechanism.

There is a strong effect of diet on the carriage of RDEC and stress, cold, dehydration or respiratory diseases can lead to the proliferation of E. coli in the rabbit caecum. Also other microbial intestinal infections can increase the E. coli growth. Following attachment by the RDEC, there is effacement of the brush border cells. This detroys the absorptive cells and the villi atrophy, there follows an inability to absorb nutrients and leads to diarrhoea. Throughout the time of infection up to 109 RDEC per gram of faeces can be excreted, resulting in an easy spead of the pathogen throughout the colony.

In the suckling rabbits, the EPEC colonise the whole large and small intestine. Previous colonisation will protect animals and milk containing IgA from protected animals will prevent attachment and the development of clinical signs in experimentally infected pups.

Generally during an outbreak the early litters effected are most severely effected, with later litters far less effected. Yellow diarrhoea in rabbits aged 3-12 days is a common feature. 100% mortality can be noted in one to two days. Weaned rabbits are usually affected one to two weeks after weaning. They stop feeding and taking water and produce watery sometimes mucoid faeces, which do not contain blood. Mortality is comparatively low at only about 10%, but with some very virulent strains it can attain 50%. The large intestine is mainly involved.

© Copyright Microbionet.

All literary matter in Microbionet is covered by copyright, and must not be reproduced, stored in a retrieval system, or transmitted in any form by electronic or mechanical means, photocopying, or recording, without written permission. This page is to be read in conjunction with the Disclaimer.

Use of this site signifes your agreement to the Legal Notices. Legal Notices © Copyright Microbionet. All rights reserved.