Potential immunosuppressive effects of Escherichia coli O157:H7 experimental infection on the bovine host.

BACKGROUND: Enterohaemorrhagic Escherichia coli (EHEC), like E. coli O157:H7 are frequently detected in bovine faecal samples at slaughter. Cattle do not show clinical symptoms upon infection, but for humans the consequences after consuming contaminated beef can be severe. The immune response against EHEC in cattle cannot always clear the infection as persistent colonization and shedding in infected animals over a period of months often occurs. In previous infection trials, we observed a primary immune response after infection which was unable to protect cattle from re-infection. These results may reflect a suppression of certain immune pathways, making cattle more prone to persistent colonization after re-infection. To test this, RNA-Seq was used for transcriptome analysis of recto-anal junction tissue and ileal Peyer's patches in nine Holstein-Friesian calves in response to a primary and secondary Escherichia coli O157:H7 infection with the Shiga toxin (Stx) negative NCTC12900 strain. Non-infected calves served as controls. RESULTS: In tissue of the recto-anal junction, only 15 genes were found to be significantly affected by a first infection compared to 1159 genes in the ileal Peyer's patches. Whereas, re-infection significantly changed the expression of 10 and 17 genes in the recto-anal junction tissue and the Peyer's patches, respectively. A significant downregulation of 69 immunostimulatory genes and a significant upregulation of seven immune suppressing genes was observed. CONCLUSIONS: Although the recto-anal junction is a major site of colonization, this area does not seem to be modulated upon infection to the same extent as ileal Peyer's patches as the changes in gene expression were remarkably higher in the ileal Peyer's patches than in the recto-anal junction during a primary but not a secondary infection. We can conclude that the main effect on the transcriptome was immunosuppression by E. coli O157:H7 (Stx-) due to an upregulation of immune suppressive effects (7/12 genes) or a downregulation of immunostimulatory effects (69/94 genes) in the ileal Peyer's patches. These data might indicate that a primary infection promotes a re-infection with EHEC by suppressing the immune function.

Clearance of Escherichia coli O157:H7 infection in calves by rectal administration of bovine lactoferrin.

Enterohemorrhagic Escherichia coli (EHEC) strains, of which E. coli O157:H7 is the best-studied serotype, are an important group of foodborne pathogens causing severe illness in humans worldwide. The main reservoirs for EHEC are ruminants, mostly cattle, which harbor the bacteria in their intestinal tracts without showing clinical symptoms. In this study, we used bovine lactoferrin, a natural occurring bactericidal and immunomodulating protein, as an antibacterial agent against EHEC infection in cattle. Nine 3-month-old Holstein-Friesian calves were experimentally infected with EHEC (strain NCTC12900). Three animals received a daily rectal spray treatment with bovine lactoferrin, three animals received an oral treatment, and three animals served as a control group. Blood samples were collected weekly and fecal samples twice weekly to monitor antibody responses and fecal excretion, respectively. Animals in the rectal group ceased shedding within 26 days of the experimental treatment and remained negative. This beneficial effect of bovine lactoferrin was not observed in the oral group, where animals were still shedding at the time of euthanasia (day 61). All groups developed serum responses, but no clear differences could be observed between the groups. However, the results indicate that the use of bovine lactoferrin as a rectal treatment can be a useful strategy to preclude further transmission of EHEC infections from cattle to humans.

Development of a Chlamydia suis-specific antibody enzyme-linked immunosorbent assay based on the use of a B-cell epitope of the polymorphic membrane protein C.

Chlamydia suis infections lead to economic loss in the pork industry. Chlamydia suis infections could be successfully treated with tetracyclines until the appearance of a tetracycline resistant phenotype, which was acquired via horizontal gene transfer of the tet(C) gene. Given the importance of C. suis as a swine pathogen and as a recently emerged tetracycline resistant pathogen with zoonotic potential, our aim was to develop a sensitive C. suis-specific antibody ELISA based on the polymorphic membrane proteins (Pmps). Chlamydia Pmps are important virulence factors and candidate antigens for serodiagnosis. We identified nine Pmps (PmpA to I) in C. suis strain MD56 using a recently developed Hidden-Markov model. PmpC was the most promising candidate for the development of a C. suis-specific antibody ELISA as the protein was absent in C. abortus, C. pecorum and C. psittaci which also infect pigs and as the protein contained C. suis-specific amino acid regions, absent in C. trachomatis PmpC. We identified an immunodominant B-cell epitope in C. suis PmpC using experimental porcine sera. The sensitivity and specificity of the PmpC ELISA was compared to the complement fixation test (CFT) and to a recombinant MOMP ELISA using experimental sera. The PmpC ELISA detected all positive control sera and was in contrast to CFT and the rMOMP ELISA 100% C. suis specific as positive control sera against other Chlamydia species did not react in the PmpC ELISA. The test was successfully validated using slaughterhouse sera and sera from clinically affected pigs. The PmpC ELISA could assist in diminishing the spread of C. suis infections in the pork industry.

In vitro and in vivo studies on the antimicrobial effect of lactoferrin against Escherichia coli O157:H7.

Escherichia coli O157:H7 shed by clinically healthy ruminants has been linked to hemorrhagic colitis and the hemolytic uremic syndrome in humans. The bacteria are spread mainly by contaminated food and water, contact with animals carrying the organisms, and person-to-person contact. Although many intervention strategies have been studied to reduce E. coli O157:H7 carriage in ruminants and its spread into the environment, none of the available methods can completely eliminate the infection. Therefore, there is need for new intervention strategies which will effectively reduce E. coli O157:H7 prevalence. Lactoferrin, a member of the transferrin protein family, is an iron-binding glycoprotein that is found in many exocrine secretions, including milk, tears, saliva, and serum. Lactoferrin has a number of biological functions including antimicrobial and immunomodulatory effects. This review summarizes latest data on the antimicrobial effect of lactoferrin against E. coli O157:H7 in in vitro and in vivo studies.

Effect of lactoferrin on release and bioactivity of Shiga toxins from different Escherichia coli O157:H7 strains.

Prevention of enterohemorrhagic Escherichia coli (EHEC) O157:H7 infections and of their severe clinical sequelae in humans remain to be a current challenge. Administration of bovine lactoferrin (bLF) proved to be effective in clearing EHEC from the bovine intestine, an important EHEC reservoir, suggesting that bLF may also be beneficial in human application against EHEC infections. To estimate the biological safety of this approach, we analyzed the effects of bLF on the main EHEC virulence factor, Shiga toxin (Stx). We quantified the release of Stx 1 and 2 from two O157:H7 EHEC strains (Stx1+Stx2+ and Stx2+ producing, respectively) cultured in the presence of bLF using ELISA assays and assessed cytotoxic effects of bLF and co-cultured EHEC on Vero cells. Effects of bLF on the stability of Stx2 were investigated using western blotting. ELISA results indicate a bLF concentration-dependent decrease of active, cell-free Stx2, but not Stx1 in EHEC cultures. High concentrations (100 and 50mg/ml) of bLF resulted in significantly reduced (p<0.05) metabolic activity rates of Vero cells, whereas a concentration of 10mg/ml bLF was considered non-toxic for Vero cells. At concentrations of 1 or 0.1mg/ml, bLF mitigated the verocytotoxicity of EHEC strains in a co-culture model up to 48h after inoculation. When only colonizing bacteria were taken into account, cytotoxicity could be significantly reduced by 10 and 1mg/ml bLF during 48h. This effect of bLF at least partly results from degradation of the Stx2 receptor-binding B-subunit.