Alexander Corbishley

Optimizing the protection of cattle against Escherichia coli O157:H7 colonization through immunization with different combinations of H7 Flagellin, Tir, Intimin-531 or EspA

Enterohemorrhagic Escherichia coli (EHEC) are important human pathogens, causing hemorrhagic colitis and hemolytic uraemic syndrome in humans. E. coli O157:H7 is the most common serotype associated with EHEC infections worldwide, although other non-O157 serotypes cause life-threatening infections. Cattle are a main reservoir of EHEC and intervention strategies aimed at limiting EHEC excretion from cattle are predicted to lower the risk of human infection. We have previously shown that immunization of calves with recombinant versions of the type III secretion system (T3SS)-associated proteins EspA, intimin and Tir from EHEC O157:H7 significantly reduced shedding of EHEC O157 from experimentally-colonized calves, and that protection could be augmented by the addition of H7 flagellin to the vaccine formulation. The main aim of the present study was to optimize our current EHEC O157 subunit vaccine formulations by identifying the key combinations of these antigens required for protection. A secondary aim was to determine if vaccine-induced antibody responses exhibited cross-reactive potential with antigens from other EHEC serotypes. Immunization with EspA, intimin and Tir resulted in a reduction in mean EHEC O157 shedding following challenge, but not the mean proportion of calves colonized. Removal of Tir resulted in more prolonged shedding compared with all other groups, whereas replacement of Tir with H7 flagellin resulted in the highest levels of protection, both in terms of reducing both mean EHEC O157 shedding and the proportion of colonized calves. Immunization of calves with recombinant EHEC O157 EspA, intimin and Tir resulted in the generation of antibodies capable of cross-reacting with antigens from non-O157 EHEC serotypes, suggesting that immunization with these antigens may provide a degree of cross-protection against other EHEC serotypes. Further studies are now required to test the efficacy of these vaccines in the field, and to formally test the cross-protective potential of the vaccines against other non-O157 EHEC.

Strain-Dependent Cellular Immune Responses in Cattle following Escherichia coli O157:H7 Colonization

ABSTRACT Enterohemorrhagic Escherichia coli (EHEC) O157:H7 causes hemorrhagic diarrhea and potentially fatal renal failure in humans. Ruminants are considered to be the primary reservoir for human infection. Vaccines that reduce shedding in cattle are only partially protective, and their underlying protective mechanisms are unknown. Studies investigating the response of cattle to colonization generally focus on humoral immunity, leaving the role of cellular immunity unclear. To inform future vaccine development, we studied the cellular immune responses of cattle during EHEC O157:H7 colonization. Calves were challenged either with a phage type 21/28 (PT21/28) strain possessing the Shiga toxin 2a (Stx2a) and Stx2c genes or with a PT32 strain possessing the Stx2c gene only. T-helper cell-associated transcripts at the terminal rectum were analyzed by reverse transcription-quantitative PCR (RT-qPCR). Induction of gamma interferon (IFN-γ) and T-bet was observed with peak expression of both genes at 7 days in PT32-challenged calves, while upregulation was delayed, peaking at 21 days, in PT21/28-challenged calves. Cells isolated from gastrointestinal lymph nodes demonstrated antigen-specific proliferation and IFN-γ release in response to type III secreted proteins (T3SPs); however, responsiveness was suppressed in cells isolated from PT32-challenged calves. Lymph node cells showed increased expression of the proliferation marker Ki67 in CD4+ T cells from PT21/28-challenged calves, NK cells from PT32-challenged calves, and CD8+ and γδ T cells from both PT21/28- and PT32-challenged calves following ex vivo restimulation with T3SPs. This study demonstrates that cattle mount cellular immune responses during colonization with EHEC O157:H7, the temporality of which is strain dependent, with further evidence of strain-specific immunomodulation.

Functional analysis of bovine TLR5 and association with IgA responses of cattle following systemic immunisation with H7 flagella

Flagellin subunits are important inducers of host immune responses through activation of TLR5 when extracellular and the inflammasome if cytosolic. Our previous work demonstrated that systemic immunization of cattle with flagella generates systemic and mucosal IgA responses. The IgA response in mice is TLR5-dependent and TLR5 can impact on the general magnitude of the adaptive response. However, due to sequence differences between bovine and human/murine TLR5 sequences, it is not clear whether bovine TLR5 (bTLR5) is able to stimulate an inflammatory response following interaction with flagellin. To address this we have examined the innate responses of both human and bovine cells containing bTLR5 to H7 flagellin from E. coli O157:H7. Both HEK293 (human origin) and embryonic bovine lung (EBL) cells transfected with bTLR5 responded to addition of H7 flagellin compared to non-transfected controls. Responses were significantly reduced when mutations were introduced into the TLR5-binding regions of H7 flagellin, including an R90T substitution. In bovine primary macrophages, flagellin-stimulated CXCL8 mRNA and secreted protein levels were significantly reduced when TLR5 transcript levels were suppressed by specific siRNAs and stimulation was reduced with the R90T-H7 variant. While these results indicate that the bTLR5 sequence produces a functional flagellin-recognition receptor, cattle immunized with R90T-H7 flagella also demonstrated systemic IgA responses to the flagellin in comparison to adjuvant only controls. This presumably either reflects our findings that R90T-H7 still activates bTLR5, albeit with reduced efficiency compared to WT H7 flagellin, or that other flagellin recognition pathways may play a role in this mucosal response.

Identification of epitopes recognised by mucosal CD4+ T-cell populations from cattle experimentally colonised with Escherichia coli O157:H7

Vaccines targeting enterohaemorrhagic Escherichia coli (EHEC) O157:H7 shedding in cattle are only partially protective. The correlates of protection of these vaccines are unknown, but it is probable that they reduce bacterial adherence at the mucosal surface via the induction of blocking antibodies. Recent studies have indicated a role for cellular immunity in cattle during colonisation, providing an impetus to understand the bacterial epitopes recognised during this response. This study mapped the epitopes of 16 EHEC O157:H7 proteins recognised by rectal lymph node CD4+ T-cells from calves colonised with Shiga toxin producing EHEC O157:H7 strains. 20 CD4+ T-cell epitopes specific to E. coli from 7 of the proteins were identified. The highly conserved N-terminal region of Intimin, including the signal peptide, was consistently recognised by mucosal CD4+ T-cell populations from multiple animals of different major histocompatibility complex class II haplotypes. These T-cell epitopes are missing from many Intimin constructs used in published vaccine trials, but are relatively conserved across a range of EHEC serotypes, offering the potential to develop cross protective vaccines. Antibodies recognising H7 flagellin have been consistently identified in colonised calves; however CD4+ T-cell epitopes from H7 flagellin were not identified in this study, suggesting that H7 flagellin may act as a T-cell independent antigen. This is the first time that the epitopes recognised by CD4+ T-cells following colonisation with an attaching and effacing pathogen have been characterised in any species. The findings have implications for the design of antigens used in the next generation of EHEC O157:H7 vaccines.

Successful treatment of a multidrug resistant Pseudomonas aeruginosa infection following a digit amputation in a Belgian Blue calf

Digit amputation is a commonly performed surgery in cattle practice. The most frequent reason for digit amputation is septic pedal arthritis, but other indications include severe trauma, pedal osteitis, non-healing sole, wall or toe ulcers, osteomyelitis of P2, septic tenosynovitis of the distal digital flexor tendons and other infectious processes of the deep digital structures. The procedure is considered a salvage procedure, but good rates of success are reported. The size of the patient is one of the key determinants of prognosis, with reported success rates varying between bulls, cows and calves. Reported complications include haemorrhage, avascular necrosis of the phalangeal fragments, wound infection and fracture of the partner claw. In this case report the authors describe digit amputation under regional and general anaesthesia for the treatment of proximal interphalangeal septic arthritis and the subsequent wound management in the face of a multidrug resistant Pseudomonas aeruginosa infection.

An outbreak of infectious bovine rhinotracheitis (IBR) in a herd vaccinated with a live glycoprotein E deleted (marker) bovine herpesvirus 1 (BoHV-1) vaccine: lessons to be learned

Vaccines are commonly used in the control of bovine respiratory disease; however, the field performance of these vaccines is poorly understood. We describe an outbreak of infectious bovine rhinotracheitis in a 383-animal beef finishing unit in Scotland four months after vaccination with a live glycoprotein E deleted (marker) bovine herpesvirus 1 (BoHV-1) vaccine. Seroconversion to the vaccine was confirmed in acute sera, and seroconversion to field virus was confirmed in convalescent sera. BoHV-1 was also identified in bronchoalveolar lavage fluid and conjunctival swabs using PCR. This outbreak highlights the importance of the reporting of veterinary vaccine suspected lack of expected efficacy events, as well as the paucity of data available to practitioners relating to the field performance of veterinary vaccines.

Quantitative analysis of antibiotic usage in British sheep flocks

The aim of this study was to examine the variation in antibiotic usage between 207 commercial sheep flocks using their veterinary practice prescribing records. Mean and median prescribed mass per population corrected unit (mg/PCU) was 11.38 and 5.95, respectively and closely correlated with animal defined daily dose (ADDD) 1.47 (mean), 0.74 (median) (R2=0.84, P<0.001). This is low in comparison with the suggested target (an average across all the UK livestock sectors) of 50 mg/PCU. In total, 80 per cent of all antibiotic usage occurred in the 39 per cent of flocks where per animal usage was greater than 9.0 mg/PCU. Parenteral antibiotics, principally oxytetracycline, represented 82 per cent of the total prescribed mass, 65.5 per cent of antibiotics (mg/PCU) were prescribed for the treatment of lameness. Oral antibiotics were prescribed to 49 per cent of flocks, 64 per cent of predicted lamb crop/farm. Lowland flocks were prescribed significantly more antibiotics than hill flocks. Variance partitioning apportioned 79 per cent of variation in total antibiotic usage (mg/PCU) to the farm level and 21 per cent to the veterinary practice indicating that veterinary practices have a substantial impact on overall antimicrobial usage. Reducing antibiotic usage in the sheep sector should be possible with better understanding of the drivers of high usage in individual flocks and of veterinary prescribing practices.

BVA's property review

I WOULD like to draw attention to a letter posted on the BVA Community recently, regarding the proposed sale of the BVAs Mansfield Street headquarters. The authors of the letter assert that ‘Mansfield Street is BVAs single most valuable asset’; however, I would contend that the BVAs single most valuable asset is, in fact, …

Adding value for farmers through herd health plans

I WAS worried by the suggestion in the discussion of the paper by Hall and Wapenaar (2012) that farmers might be being seen as ‘unconscious[ly] incompetent’ with regard to their appreciation of the benefits of herd health and production management (HH&PM). While this may be true for a minority of dairy farmers, and perhaps a greater number of beef and lamb producers, it is not my experience of the progressive dairy client. Have the authors considered the alternative idea, that it is the veterinary …