A. D. Wales

Attaching-effacing bacteria in animals

n Summaryn n Enteric bacteria with a demonstrable or potential ability to form attaching-effacing lesions, so-called attaching-effacing (AE) bacteria, have been found in the intestinal tracts of a wide variety of warm-blooded animal species, including man. In some host species, for example cattle, pigs, rabbits and human beings, attaching-effacing Escherichia coli (AEEC) have an established role as enteropathogens. In other host species, AE bacteria are of less certain significance. With continuing advances in the detection and typing of AE strains, the importance of these bacteria for many hosts is likely to become clearer. The pathogenic effects of AE bacteria result from adhesion to the intestinal mucosa by a variety of mechanisms, culminating in the formation of the characteristic intimate adhesion of the AE lesion. The ability to induce AE lesions is mediated by the co-ordinated expression of some 40 bacterial genes organized within a so-called pathogenicity island, known as the “Locus for Enterocyte Effacement”. It is also believed that the production of bacterial toxins, principally Vero toxins, is a significant virulence factor for some AEEC strains. Recent areas of research into AE bacteria include: the use of Citrobacter rodentium to model human AEEC disease; quorum-sensing mechanisms used by AEEC to modulate virulence gene expression; and the potential role of adhesion in the persistent colonization of the intestine by AE bacteria. This review of AE bacteria covers their molecular biology, their occurrence in various animal species, and the diagnosis, pathology and clinical aspects of animal diseases with which they are associated. Reference is made to human pathogens where appropriate. The focus is mainly on natural colonization and disease, but complementary experimental data are also included.n n

Non-toxigenic Escherichia coli O157:H7 strain NCTC12900 causes attaching-effacing lesions and eae-dependent persistence in weaned sheep

Ruminants are regarded as a primary reservoir for Escherichia coli O157:H7, an important human pathogen. Intimin, encoded by the Locus of Enterocyte Effacement by E. coli O157:H7 organisms, has been cited as one bacterial mechanism of colonisation of the gastrointestinal tract. To confirm this and to test whether a non-toxigenic E. coli O157:H7 strain would colonise and persist in a sheep model, E. coli O157:H7 strain NCTC12900, that lacks Shiga toxin (stx) genes, was evaluated for use in a sheep model of persistence. Following oral inoculation of six-week-old sheep, persistent excretion of NCTC12900 was observed for up to 48 days. E. coli O157-associated attaching-effacing (AE) lesions were detected in the caecum and rectum of one six-week-old lamb, one day after inoculation. This is the first recorded observation of AE lesions in orally inoculated weaned sheep. Also, mean faecal excretion scores of NCTC12900 and an isogenic intimin (eae)-deficient mutant were determined from twenty-four six-week-old orally inoculated sheep. The eae mutant was cleared within 20 days and had lower mean excretion scores at all time points after day one post inoculation compared with the parental strain that was still being excreted at 48 days. Tissues were collected post mortem from animals selected at random from the study groups over the time course of the experiment. The eae mutant was detected in only 1/43 samples but the parental strain was recovered from 64/140 samples primarily from the large bowel although rumen, duodenum, jejunum, and ileum were culture positive especially from animals that were still excreting at and beyond 27 days after inoculation.

Attaching and effacing lesions in the intestines of two calves associated with natural infection with Escherichia coli O26:H11.

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Experimental infection of six-month-old sheep with Escherichia coli O157:H7

ENTEROHAEMORRHAGIC Escherichia coli (EHEC) serotype 0157:H7 infection in humans is potentially fatal in the very young and the elderly, and major incidents worldwide have been well documented (Smith and Scotland 1993, Boyce and others 1995, Swinbanks 1996). A distinctive gastrointestinal illness of severe blood diarrhoea (haemorrhagic colitis) typically without fever (Riley and others 1983), may be followed by acute renal failure, thrombocytopenia and microangiopathic haemolytic anaemia (haemorrhagic uraemic syndrome) (Karmali and others 1983). Molecular pathogenesis studies have demonstrated roles in the disease process for toxins, an enterohaemolysin, the intimin adherence factor and possibly the pO157 plasmid and lipopolysaccharide (Nataro and Kaper 1998). EHEC 0157:H7 is present in the faecal flora of cattle and sheep (Griffin and Tauxe 1991). Cattle may be readily infected both naturally and experimentally, exhibiting mild transient diarrhoea in calves and an asymptomatic transient carrier status in older animals (Cray and Moon 1995, Brown and others 1997, Woodward and others 1999, Wray and oth-

Efficacy of a Live Attenuated Escherichia coli O78∶K80 Vaccine in Chickens and Turkeys

SUMMARY A candidate live vaccine for avian pathogenic Escherichia coli (APEC) was constructed from a virulent field APEC O78 strain by mutation of the aroA gene. The mutant was highly similar to the parent wild-type strain in respect of colony morphology, motility, growth in suspension, hemagglutination, Congo Red binding, HEp-2 cell adhesion, and the elaboration of surface antigens type 1 fimbriae and flagella, although production of curli fimbriae was reduced marginally. The mutant proved avirulent when inoculated into 1-day-old chicks by spray application and when presented again in the drinking water at 7 days of age. Chickens and turkeys vaccinated with an O78 aroA mutant were protected against a challenge at 6 wk of age by virulent APEC strains. RESUMEN Eficacia de una vacuna viva atenuada de Escherichia coli O78∶K80 en pollos y pavos. Se elaboró una vacuna viva para Escherichia coli patógena aviar (APEC) a partir de una cepa de campo patógena para las aves y virulenta O78 mediante mutación del gene aroA. La mutante fue muy similar a la cepa silvestre original con respecto a la morfología de la colonia, motilidad, crecimiento en suspensión, hemaglutinación, afinidad al rojo Congo, adhesión a células HEp-2 y la elaboración de antígenos de superficie tipo 1 fimbria y flagelo, aunque la producción de fimbrias rizadas se redujo marginalmente. La mutante no mostró virulencia cuando se inoculó en pollitos de un día vía aerosol, ni cuando se administró nuevamente a los 7 días de edad, vía agua de bebida. Los pollos y pavos vacunados con la mutante O78 aroA fueron protegidos contra el desafío a las 6 semanas de edad con una cepa de E. coli patógena aviar virulenta.

Attaching and effacing lesions in the intestines of an adult goat associated with natural infection with Escherichia coli O145

THERE have been few reports of attaching and effacing Escherichia coli (AEEC) infections in goats. A severe outbreak was recorded by Drolet and others (1994) affecting neonatal goat kids, of which 21 of 34 born alive died after having diarrhoea. A further case (Duhamel and others 1992) involved a goat approximately two months old, with severe diarrhoea of three weeks’ duration. This animal was part of a drug toxicity trial, which may have been an exacerbating factor. This short communication describes a naturally occurring infection with AEEC in an adult goat with diarrhoea. Nine adult diary goats out of a herd of 300 had died over the course of a month with a history of milk drop, anorexia and weakness followed by death within a few days. The 10th animal, a three-year-old Saanen cross nanny which showed similar clinical signs, was submitted to the Veterinary Laboratories Agency (VLA) – Langford for examination. The animal was depressed, remained recumbent during clinical examination and had mild diarrhoea. Blood samples were collected for routine haematological and biochemical analysis. Haematological examination indicated dehydration, with a packed-cell volume of 0·53 litre/litre (reference range 0·24 to 0·39 litre/litre). The blood urea level was 94·7 mmol/litre (reference range 4·0 to 8·6 mmol/litre) and there was a raised blood creatine kinase level of 1083 U/litre at 37°C (reference range 0 to 100 U/litre), consistent with muscle damage due to recumbency. The remaining parameters were within normal limits. The goat was euthanased by intravenous pentobarbitone injection and a postmortem examination was performed immediately. At postmortem examination the carcase weighed 77 kg and had a large amount of abdominal fat. No gross lesions were identified in the gastrointestinal tract. The rumen contained a large volume of pale straw-coloured fluid and a little roughage. The contents of the abomasum were fluid. The small and large intestines contained a moderate amount of yellow, watery fluid and the mucosa appeared normal. The rectal contents were semisolid. The kidneys appeared slightly enlarged, and the uterus contained excess clear fluid consistent with pseudopregnancy. No other abnormalities were detected. Samples of the small and large intestinal contents, liver, heart blood and brain were collected for routine bacteriological examination. Smears of small intestinal mucosa were stained using Gram’s stain. Ten colonies of E coli selected at random from sweeps of the contents taken from the large and small intestines were subcultured onto Dorset’s egg slopes for preliminary typing as described by Pearson and others (1999). Samples of the duodenum, jejunum, ileum and colon were fixed immediately in 10 per cent neutral-buffered formalin; the tissues were processed routinely to paraffin wax, and 4 μm sections were stained with haematoxylin and eosin. For peroxidase-antiperoxidase (PAP) immunostaining, additional 4 μm sections of the ileum and colon were mounted on organosilane-coated slides and incubated at room temperature with commercially available rabbit anti-O45, anti-O71 and anti-O145 antisera (Prolabs) at a dilution of 1/1000. These antisera were selected after preliminary analysis of the E coli isolates recovered from the goats. Goat anti-rabbit antiserum (Sigma), rabbit PAP (Dako) and diaminobenzidine were then applied sequentially. Sections incubated with normal rabbit serum (diluted 1/500 and 1/1000) and rabbit anti-O157 and anti-O26 antisera were used as controls. For electron microscopic studies, a small area of the ileum was cut from the paraffin wax block and processed as described by Pearson and others (1989). Bacteriological culture of the small and large intestinal contents produced a heavy, pure growth of coliforms. Salmonella species were not isolated using specific culture techniques. The 10 randomly selected coliform colonies were tested using the multiplex PCR described by Meng and others (1997). Seven isolates possessed the eae gene, encoding the 94 kDa outer membrane protein intimin, but none was positive for stx or stx2, the genes encoding Shiga-like toxins. Five of the seven eae-positive isolates were identified as E coli O145, one as E coli O45 and one as E coli O71. Following immunostaining, the five E coli O145 isolates were analysed further by the genetic methods of Cookson and others (2002) and each was found to possess genes encoding the β-intimin subtype and CNF1 and CNF2 cytotoxins. There were no significant isolates following culture of heart blood, liver or brain. Clostridia-like organisms were not identified in Gram-stained smears of the small intestinal contents. Histological examination of the duodenum and jejunum revealed moderate separation of the villous epithelium from the lamina propria, associated with early autolytic changes, but the villi were otherwise normal. In the ileum, severe stunting and fusion of villi, with flattening of the mucosa and an irregular epithelial surface, was observed (Fig 1). Adherent bacteria were present as multifocal colonies of variable size on the mucosal surface. In the colon, a few areas of focal, irregular surface epithelium were seen, some of which were associated with adherent bacteria similar to those observed in the ileum. Examination of the kidneys revealed a marked ectasia of the distal convoluted tubules, with many of them containing oxalate casts. Sparse neutrophil foci were present in a few tubules. Immunostained ileum and colon sections demonstrated bacterial expression of E coli O145 antigen (Fig 2), but E coli O45 and O71 staining was not identified. Electron microscopy of the ileum confirmed that these bacteria were associated with attachingeffacing (AE) lesions (Fig 3). Veterinary Record (2004) 155, 807-808

Grass sickness with atypical presentation in a young zebra

EQUINE grass sickness (EGS) is a dysautonomia of undetermined aetiology occurring almost exclusively in animals at pasture. It is characterised pathologically by chromatolysislike degeneration of neurones in peripheral autonomic ganglia, the enteric nervous system and certain nuclei of the brainstem (Obel 1955, Barlow 1969). The primary clinical feature is disordered alimentary tract motility usually presenting as gut stasis, with abdominal pain and reduced faecal output (Edwards 1987). Dysphagia, gastric reflux, depression, patchy sweating and muscle tremors are common features (Doxey and others 1991). Acute and chronic EGS is recognised. In addition to the domestic horse, there are reports of grass sickness in the donkey, common zebra and Przewalski horse (Ashton and others 1977). This short communication describes a case of acute grass sickness in a Chapman zebra (Equus zebra chapmani), with atypical clinical features and macroscopic pathology. An 11-month-old homebred female zebra, previously in good health and kept on permanent pasture with a group of six healthy zebras at a wildlife park in the UK, showed signs of ill health. Initially, the animal was noted to be standing apart from the group, holding its tail down, feeding less and frequently recumbent. Two days later, a visual veterinary examination of the boxed animal revealed marked ataxia in all limbs, with the hindlimbs most severely affected. There was a tendency to stagger sideways, a reluctance to move, a head tilt and depressed tail carriage. An anti-inflammatory treatment of flunixin meglumine (Finadyne Solution; Schering Plough) was administered intramuscularly. The following day (three days after the initial presentation), the zebra had collapsed and was apparently blind, with minimal menace response. No faeces had been seen and there was no evidence of spontaneous gastric reflux. Intravenous calcium borogluconate (CALC No 1; Vericore) was administered, with no clinical response, and the animal was euthanased using intravenous pentobarbi-