U. Weyer

A mixture containing galactooligosaccharide, produced by the enzymic activity of Bifidobacterium bifidum, reduces Salmonella enterica serovar Typhimurium infection in mice

The prebiotic Bimuno is a mixture containing galactooligosaccharide, produced by the galactosyltransferase activity of Bifidobacterium bifidum NCIMB 41171 in the presence of lactose. Previous studies have implicated prebiotics in reducing infections by enteric pathogens, thus it was hypothesized that Bimuno may confer some protection in the murine host from Salmonella enterica serovar Typhimurium (S. Typhimurium) infection. In this study, infection caused by S. Typhimurium SL1344nal(r) in the presence or absence of Bimuno was assessed using tissue culture assays, a murine ligated ileal gut loop model and a murine oral challenge model. In tissue culture adherence and invasion assays with HT-29-16E cells, the presence of approximately 2 mM Bimuno significantly reduced the invasion of S. Typhimurium SL1344nal(r) (P<0.0001). In the murine ligated ileal gut loops, the presence of Bimuno prevented colonization and the associated pathology of S. Typhimurium. In the BALB/c mouse model, the oral delivery of Bimuno prior to challenge with S. Typhimurium resulted in significant reductions in colonization in the five organs sampled, with highly significant reductions being observed in the spleen at 72 and 96 h post-challenge (P=0.0002, <0.0001, respectively). Collectively, the results indicate that Bimuno significantly reduced the colonization and pathology associated with S. Typhimurium infection in a murine model system, possibly by reducing the invasion of the pathogen into host cells.

Purified galactooligosaccharide, derived from a mixture produced by the enzymic activity of Bifidobacterium bifidum, reduces Salmonella enterica serovar Typhimurium adhesion and invasion in vitro and in vivo.

The prebiotic Bimuno(®) is a mixture containing galactooligosaccharides (GOSs), produced by the galactosyltransferase activity of Bifidobacterium bifidum NCIMB 41171 using lactose as the substrate. Previous in vivo and in vitro studies demonstrating the efficacy of Bimuno(®) in reducing Salmonella enterica serovar Typhimurium (S. Typhimurium) colonization did not ascertain whether or not the protective effects could be attributed to the prebiotic component GOS. Here we wished to test the hypothesis that GOS, derived from Bimuno(®), may confer the direct anti-invasive and protective effects of Bimuno(®). In this study the efficacy of Bimuno(®), a basal solution of Bimuno(®) without GOS [which contained glucose, galactose, lactose, maltodextrin and gum arabic in the same relative proportions (w/w) as they are found in Bimuno(®)] and purified GOS to reduce S. Typhimurium adhesion and invasion was assessed using a series of in vitro and in vivo models. The novel use of three dimensionally cultured HT-29-16E cells to study prebiotics in vitro demonstrated that the presence of ∼ 5 mg Bimuno(®) ml(-1) or ∼ 2.5 mg GOS ml(-1) significantly reduced the invasion of S. Typhimurium (SL1344nal(r)) (P<0.0001). Furthermore, ∼ 2.5 mg GOS ml(-1) significantly reduced the adherence of S. Typhimurium (SL1344nal(r)) (P<0.0001). It was demonstrated that cells produced using this system formed multi-layered aggregates of cells that displayed excellent formation of brush borders and tight junctions. In the murine ligated ileal gut loops, the presence of Bimuno(®) or GOS prevented the adherence or invasion of S. Typhimurium to enterocytes, and thus reduced its associated pathology. This protection appeared to correlate with significant reductions in the neutral and acidic mucins detected in goblet cells, possibly as a consequence of stimulating the cells to secrete the mucin into the lumen. In all assays, Bimuno(®) without GOS conferred no such protection, indicating that the basal solution confers no protective effects against S. Typhimurium. Collectively, the studies presented here clearly indicate that the protective effects conferred by Bimuno(®) can be attributed to GOS.

Protection of Eurasian badgers (Meles meles) from tuberculosis after intra-muscular vaccination with different doses of BCG

Mycobacterium bovis infection is widespread in Eurasian badger (Meles meles) populations in Great Britain and the Republic of Ireland where they act as a wildlife reservoir of infection for cattle. Removal of infected badgers can significantly reduce the incidence of bovine tuberculosis (TB) in local cattle herds. However, control measures based on culling of native wildlife are contentious and may even be detrimental to disease control. Vaccinating badgers with bacillus Calmette-Guerin (BCG) has been shown to be efficacious against experimentally induced TB of badgers when administered subcutaneously and orally. Vaccination may be an alternative or complementary strategy to other disease control measures. As the subcutaneous route is impractical for vaccinating wild badgers and an oral vaccine bait formulation is currently unavailable, we evaluated the intramuscular (IM) route of BCG administration. It has been demonstrated that the IM route is safe in badgers. IM administration has the practical advantage of being relatively easy to perform on trapped wild badgers without recourse to chemical immobilisation. We report the evaluation of the efficacy of IM administration of BCG Danish strain 1331 at two different doses: the dose prescribed for adult humans (2-8×10(5)colony forming units) and a 10-fold higher dose. Vaccination generated a dose-dependent cell-mediated immune response characterised by the production of interferon-γ (IFNγ) and protection against endobronchial challenge with virulent M. bovis. Protection, expressed in terms of a significant reduction in the severity of disease, the number of tissues containing acid-fast bacilli, and reduced bacterial excretion was statistically significant with the higher dose only.

Intermittent Escherichia coli O157:H7 colonisation at the terminal rectum mucosa of conventionally-reared lambs.

In cattle, the lymphoid rich regions of the rectal-anal mucosa at the terminal rectum are the preferred site for Escherichia coli O157:H7 colonisation. All cattle infected by rectal swab administration demonstrate long-term E. coli O157:H7 colonisation, whereas orally challenged cattle do not demonstrate long-term E. coli O157:H7 colonisation in all animals. Oral, but not rectal challenge of sheep with E. coli O157:H7 has been reported, but an exact site for colonisation in sheep is unknown. To determine if E. coli O157:H7 can effectively colonise the ovine terminal rectum, in vitro organ culture (IVOC) was initiated. Albeit sparsely, large, densely packed E. coli O157:H7 micro-colonies were observed on the mucosa of ovine and control bovine terminal rectum explants. After necropsy of orally inoculated lambs, bacterial enumeration of the proximal and distal gastrointestinal tract did suggest a preference for E. coli O157:H7 colonisation at the ovine terminal rectum, albeit for both lymphoid rich and non-lymphoid sites. As reported for cattle, rectal inoculation studies were then conducted to determine if all lambs would demonstrate persistent colonisation at the terminal rectum. After necropsy of E. coli O157:H7 rectally inoculated lambs, most animals were not colonised at gastrointestinal sites proximal to the rectum, however, large densely packed micro-colonies of E. coli O157:H7 were observed on the ovine terminal rectum mucosa. Nevertheless, at the end point of the study (day 14), only one lamb had E. coli O157:H7 micro-colonies associated with the terminal rectum mucosa. A comparison of E. coli O157:H7 shedding yielded a similar pattern of persistence between rectally and orally inoculated lambs. The inability of E. coli O157:H7 to effectively colonise the terminal rectum mucosa of all rectally inoculated sheep in the long term, suggests that E. coli O157:H7 may colonise this site, but less effectively than reported previously for cattle.

The effect of oral vaccination with Mycobacterium bovis BCG on the development of tuberculosis in captive European badgers (Meles meles)

The European badger (Meles meles) is a reservoir host of Mycobacterium bovis and responsible for a proportion of the tuberculosis (TB) cases seen in cattle in the United Kingdom and Republic of Ireland. An injectable preparation of the bacillus Calmette-Guérin (BCG) vaccine is licensed for use in badgers in the UK and its use forms part of the bovine TB eradication plans of England and Wales. However, there are practical limitations to the widespread application of an injectable vaccine for badgers and a research priority is the development of an oral vaccine deliverable to badgers in bait. Previous studies reported the successful vaccination of badgers with oral preparations of 108 colony forming units (CFU) of both Pasteur and Danish strains of BCG contained within a lipid matrix composed of triglycerides of fatty acids. Protection against TB in these studies was expressed as a reduction in the number and apparent progression of visible lesions, and reductions in the bacterial load and dissemination of infection. To reduce the cost of an oral vaccine and reduce the potential for environmental contamination with BCG, it is necessary to define the minimal efficacious dose of oral BCG for badgers. The objectives of the two studies reported here were to compare the efficacy of BCG Danish strain in a lipid matrix with unformulated BCG given orally, and to evaluate the efficacy of BCG Danish in a lipid matrix at a 10-fold lower dose than previously evaluated in badgers. In the first study, both BCG unformulated and in a lipid matrix reduced the number and apparent progression of visible lesions and the dissemination of infection from the lung. In the second study, vaccination with BCG in the lipid matrix at a 10-fold lower dose produced a similar outcome, but with greater intra-group variability than seen with the higher dose in the first study. Further research is needed before we are able to recommend a final dose of BCG for oral vaccination of badgers against TB or to know whether oral vaccination of wild badgers with BCG will significantly reduce transmission of the disease.

Interaction of enterohemorrhagic Escherichia coli O157:H7 with mouse intestinal mucosa

In this study, we used mouse ileal loops to investigate the interaction of enterohemorrhagic Escherichia coli (EHEC) O157:H7 with the mouse intestinal mucosa. With a dose of 10(9) and 3 h incubation, EHEC O157 was detected in the lumen and to a lesser extent associated with the epithelium. Typical attaching and effacing (A/E) lesions were seen, albeit infrequently. While the effector protein Tir was essential for A/E lesion formation, the bacterial type III secretion system adaptor protein TccP was dispensable. These results suggest that A/E lesions on mouse intestinal mucosa can be formed independently of robust actin polymerization.

Failure to detect transmission of Escherichia coli O157:H7 from orally dosed nannies to sucking kids at foot

INFECTION caused by Escherichia coli serotype O157:H7 was first recognised to be associated with haemorrhagic colitis, haemolytic uraemic syndrome and thrombocytopenic purpura in human beings in the early 1980s. E coli O157:H7 is now classified as belonging to the recently defined enterohaemorrhagic E coli pathotype. This pathotype is recognised worldwide as being the leading cause of both haemorrhagic colitis and haemolytic uraemic syndrome (Nataro and Kaper 1998). Transmission of E coli O157:H7 is via the oral-faecal route (Pepin and others 1997, Locking and others 2001), with cattle and sheep being recognised as major reservoirs of the bacterium (Griffin and Tauxe 1991, Chapman and others 1997). Other reservoirs include feral animals, rabbits and gulls, among other species (Griffin and Tauxe 1991, Pritchard and others 2000), and evidence is accumulating to suggest that goats’ milk and goats are also potential sources. Shukla and others (1995) reported cases of bloody diarrhoea in human beings that were traced to a farm visitor centre in Leicestershire. Strains of E coli O157:H7 phage type (PT) 2 with identical restriction fragment length polymorphism patterns were isolated from these cases and from six goats and four cattle at the centre. Three other incidents in the UK have implicated goats on visitor farms as being potential sources of infection (Chapman and others 2000, Pritchard and others 2000, Payne and others 2003). In Europe, Heuvelink and others (2002) reported that two of 11 petting zoos that had goats were positive for E coli O157:H7. In Bohemia, Bielaszewska and others (1997) detected identical isolates of E coli O157:H7 PT2 in four cases of haemolytic uraemic syndrome in children, an asymptomatic human carrier who drank goats’ milk, the source goat and unpasteurised goats’ milk. An outbreak of E coli O157:H7 associated with unpasteurised goats’ milk has also been described in Canada (McIntyre and others 2002), while in the Bergamo region of Italy, Foschino and others (2002) reported a 1·7 per cent incidence rate of E coli O157:H7 in samples of raw goats’ milk intended for cheese making, although it was not associated with infection in human beings. Despite these incidents involving goats as a source of E coli O157:H7, the biology of E coli O157:H7 in this host is not properly understood. In addition, little is known of the prevalence of E coli O157:H7 in goats, with only a few reports suggesting very low prevalence rates in the region of 0·2 per cent (Cid and others 2001, de la Fuente and others 2002, Blanco and others 2003, Dontorou and others 2004). Wales and others (2005) showed that six-day-old conventional kids may be colonised by E coli O157:H7, and small attaching and effacing (AE) lesions induced by E coli O157:H7 in the mucosa of the large intestine were observed after deliberate oral inoculation. While these data suggest that kid goats are susceptible to colonisation by E coli O157:H7, it is unclear whether older goats are also susceptible to colonisation by E coli O157:H7 and whether transmission from affected nannies to presumed susceptible neonate kids at foot is a risk factor in the management of goats on petting zoos and farms. This short communication describes an experiment in which nannies with kids at foot were inoculated orally with E coli O157:H7 to determine the course of the dosed organisms. All animal procedures were approved following ethical review at the Veterinary Laboratories Agency, and complied with the Animals (Scientific Procedures) Act 1986; the procedures were performed under Home Office licence 70/5722. Previous studies in another small ruminant model, lambs and sheep, made use of E coli O157:H7 strain NCTC 12900 Nalr. This strain does not produce verocytotoxins and can be used at containment level II; it also generates a localised adherence phenotype on cultured HEp-2 and bovine primary epithelial cells with actin rearrangements characteristic of the AE lesion (Dibb-Fuller and others 2001, Cookson and Woodward 2003). Furthermore, this strain forms AE lesions in the large intestine of orally inoculated six-day-old goats (Wales and others 2005), in the large intestine of six-week-old lambs (Wales and others 2001b) and on the mucosa in ligated large intestinal loops in six-month-old lambs (Wales and others 2002). Additionally, it persists in six-week-old and six-monthold conventional lambs (Cookson and others 2002, Wales and others 2001a, 2005, Woodward and others 2003). NCTC 12900 Nalr was inoculated into 100 ml aliquots of Luria-Bertani broth in 250 ml conical flasks. After incubation for 16 hours at 37°C with gentle agitation, the bacterial cells were harvested by centrifugation at 3000 g for 10 minutes, and resuspended to one-fifth of the original volume in phosphatebuffered saline. The bacterial suspensions contained approximately 1 x 109colony-forming units (cfu)/ml, as determined by serial dilution and plating, and 10 ml of this was used as the oral inoculum for each animal. Five pregnant nannies (goats 1 to 5), approximately five years of age, were housed indoors and provided with standard rations and water ad libitum. Rectal swabs were taken before parturition and treated by immunomagnetic separation and plating on cefixime-tellurite sorbitol MacConkey (CT-SMAC) agar as described by Wales and others (2001a, b) in order to detect E coli O157:H7. None of the nannies was positive for E coli O157:H7, but all harboured sorbitol-fermenting commensal E coli. After parturition, the kids remained with their nanny and sucked. Rectal swabs from each of the kids were taken on day 6, after which each nanny was challenged with E coli O157:H7 strain NCTC 12900 Nalr prepared as described above. No kid was Veterinary Record (2005) 157, 659-661

Pelioid hepatocellular carcinoma in an adult Eurasian badger (Meles meles).

A mass was identified within the left lateral lobe of the liver of a 10-year-old Eurasian badger (Meles meles). The mass was friable and multilobulated, with blood-filled spaces between the lobules. Microscopically, the lesion consisted of sheets and trabeculae of neoplastic hepatocytes often forming cystic spaces containing erythrocytes, fibrin and necrotic debris. The histological appearance was consistent with hepatocellular carcinoma (HCC). Immunohistochemically, the neoplastic cells expressed cytokeratin 18 but not von Willebrand factor. Multiple intranuclear (amphophilic or acidophilic) inclusion bodies were observed in hepatocytes at the junction between the tumour and normal hepatic tissue. HCCs have also been reported in other domestic and wild animals. As hepadnavirus infection has been associated with HCC in woodchucks, further histochemical and transmission electron microscopical studies were performed; however, these demonstrated that the inclusions consisted of lipid droplets and not viral particles. To our knowledge, this is the first report of a naturally occurring HCC in a Eurasian badger.

Escherichia coli O115 forms fewer attaching and effacing lesions in the ovine colon in the presence of E. coli O157:H7.

Escherichia coli O115 has been isolated from healthy sheep and was shown to be associated with attaching-effacing (AE) lesions in the large intestine. Following previous observations of interactions between E. coli O157 and O26, the aim of the present study was to assess what influence an O115 AE E. coli (AEEC) would have on E. coli O157 colonisation in vitro and in vivo. We report that E. coli O115- and O157-associated AE lesions were observed on HEp-2 cells and on the mucosa of ligated ovine spiral colon. In single strain inoculum, E. coli O115 associated intimately with HEp-2 cells and the spiral colon in greater numbers than E. coli O157:H7. However, in mixed inoculum studies, the number of E. coli O115 AE lesions was significantly reduced suggesting negative interference by E. coli O157. Use of the ligated colon model in the present work has allowed in vitro observations to be extended and confirmed whilst using a minimum of experimental animals. The findings support a hypothesis that some AEEC can inhibit adhesion of other AEEC in vivo. The mechanisms involved may prove to be of utility in the control of AE pathovars.