On-farm/pre-harvest control strategies for the reduction of Shiga-toxin producing Escherichia coli

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1 On-farm/pre-harvest control strategies for the reduction of Shiga-toxin producing Escherichia coli Dr Adrian Cookson Hopkirk Institute, Palmerston North Food Assurance & Meat Quality Team, AgResearch

2 Rationale Animal infection & colonisation Horizontal/vertical transmission Reduced on-farm STEC prevalence Human infection Product contamination Environmental contamination

3 Aim To identify and validate through on-farm studies, practical and effective STEC interventions able to be readily adopted and implemented into current/future dairy (varied) farm systems cheaply with no deleterious impact on animal health/productivity. In collaboration with AgResearch experts (food microbiology, environmental microbiology, farm systems, nutritionists, forage feeds, farm system adoption, social scientists) and external stakeholders (government, meat/dairy industry, public health and other NZFSSRC partners)

4 Focus Dairy farming operators Operations representing multiple intervention target points Pre-partum dams Young calves Weaned calves Heifer replacements Dairy herd animals

5 STEC intervention groupings Cattle water and feed Live animal treatments Management practices

6 Cattle water and feed Ryegrass/clover cultivars Trough cleaning Brassicas, chicory etc. Supplemental feeds

7 Live animal treatments Probiotics Bacteriophage therapy Vaccines

8 Management practices Calf management Heifer replacer management Feed storage On-farm biosecurity Off-pasture housing Vector control Effluent management

9 Systems approach to understanding the risk landscape

10 New Zealand issues/considerations Intervention effectiveness vs. practicality Measure of intervention effectiveness? STEC O157 Economically important STEC (O157, O26, O45, O103, O111, O121, O145) All STEC Generic E. coli STEC detection methods? Contrasting phenotypes & intervention susceptibilities Horizontal transmission Neonatal calf, weaned calf, heifer replacement, dairy cow Varying dairy farm practices Systems 1 to 5 Use of supplemental feeds (maize silage, PKE, brassicas) Housing of animals, use of HH and SOP Climate, geography, rainfall, humidity

11 Intervention adoption STEC infection of cattle No usual signs of clinical disease No loss of animal productivity No restrictions on sale or processing of animal No tangible farm level benefits to reduce STEC prevalence No increased animal productivity No premium for STEC-free animals No economic gains Farmer education and knowledge Implementation of general zoonotic mitigation programme Farmer advice for on-farm control of multiple zoonoses Identification of critical control points Mutual control points for several pathogens

12 Next steps Engage with and seek feedback on STEC intervention discussion document AgResearch and NZFSSRC collaborators Industry stakeholders Undertake trials to measure effectiveness of potential interventions on farms recognised as STEC-positive

13 Acknowledgements Funded through AgResearch Core Funded research as part of the Food Provenance & Assurance (FPA) programme. Pre-Harvest Food Chain Control component of FPA programme aims to understand and mitigate the risks associated with increased pathogen loading on intensifying dairy farm systems with the view of developing pre-harvest interventions for reducing contamination further along the food chain FAMQ Team and mepilab

14 Use of gnd to assess Escherichia coli community diversity using cultureindependent methods. Adrian L. Cookson 1,2, Angela Reynolds 1, Rose Collis 1, Patrick J. Biggs 2, Nigel P. French 2, and Gale Brightwell 1. 1 Food & Bio-based Products, AgResearch Ltd, Palmerston North, New Zealand 2 mepilab, Institute of Veterinary, Animal and Biomedical Sciences (IVABS), Massey University, Palmerston North, New Zealand

15 Project Background E. coli differentiation Serology O, H and K grouping Pathotypes EPEC, STEC, ETEC, EAEC, EIEC, ExPEC, (Nissle 1917, HS) Subtyping Pulsed field gel electrophoresis (PFGE) Multi locus sequence typing (MLST) Insertion sequence (IS) typing Genome sequencing SNP profiling E. coli differentiation using pure cultures

16 Project Background E. coli diversity Previously assessed using culture-based methods 1. Faecal samples from beef cattle fed: 1 Roughage & molasses, 30 serotypes (n=10 animals) Roughage, 21 serotypes (n=11) Grain, 17 serotypes (n=9) 2. Biotypes from human faecal samples: 2 Range of 1 to 15 (average 5) from healthy humans (n=9) over 6 weeks (range of samples/volunteer) Diversity of E. coli using culture-independent methods unknown 1 Bettelheim et al., J Appl Micro Apperloo-Renkema et al., Epi & Inf

17 Project Aims To identify a gene for barcoding E. coli populations To assess its use for community profiling of E. coli from complex matrices (e.g. bovine faecal samples)

18 Location of barcode targets Focus on hot-spots for recombination/horizontal gene transfer O antigen biosynthesis gene clusters (O-AGC) prone to recombination E.g. evolution of STEC O157 from STEC O recognised E. coli serogroups based on antigenic variability many more untypeable? Representative O-AGC sequenced 1 Development of serogroup-specific PCRs 2 1 Iguchi et al., DNA Res Iguchi et al., J Clin Microbiol

19 gnd 6-phosphogluconate dehydrogenase Housekeeping gene often associated with O-AGC in Enterobacteriaceae Third enzyme reaction of pentose phosphate pathway Described as passive hitch-hiker 1 with existing O-AGC variants Variability noted in prior work through MLEE 2, RFLP 3, sequencing 1,4 1 Nelson & Selander, PNAS Selander & Levin, Science Dykhuizen & Green, J Bact Gilmour et al., J Med Micro

20 gnd sequence analysis Alignment made of >1000 E. coli gnd DNA sequences from PATRIC, GenBank, IMG etc. Degenerate PCR primers designed for gnd amplicon sequencing (Illumina) Designed to be sequenced with good overlap on 2 x 250 bp MiSeq run gnd database created including 300 unique combinations of E. coli serotype and gnd sequence Covers all 184 serogroups and 35 untypeable or rough strains

21 Method sensitivity and specificity gnd amplicons obtained from all E. coli test cultures Separate O-AGC groups may have same gnd sequence (e.g. O17 and O44: >99.9% similar) 1 O157 gnd sequence variation differentiates STEC from non-stec 2 gnd amplicons from STEC Super Six serogroups are not always distinguishable from corresponding stx-negative strains Multiple gnd sequences associated with diverse lineages of same serogroup indicative of separate O-AGC recombination events (e.g. O91, O104, O128) 1 Iguchi et al., DNA Res Tarr et al., J Bact

22 Detailed sequencing study Animal study (n=23) to assess role of bifidobacteria on calf (3-4 days) health Treatment group orally dosed daily with 2 x bifidobacteria (14 days) RAMS and faecal samples taken from calves at days of age Barcoded gnd amplicons generated from DNA extracts Faeces (23) mtsb pre-enrichment (23) mtsb post-enrichment boiled lysate (23) mtsb post-enrichment kit (23) Synthetic libraries (4) 4-5 colonies (MAC plates)/animal MiSeq (2 x 250bp PE) analysis of gnd amplicon libraries 96 libraries undergoing analyses

23 gnd applications Identify temporal changes in community structure E.g. before/during/after interventions Identification of E. coli that may exclude STEC7 Detection of industry/clinically important strains in complex samples using culture-independent methods Targeted, culture-independent approach for E. coli isolation Alternative to serology Successfully adopted to serotype STEC

24 Acknowledgements Patrick Biggs MiSeq data analysis & bioinformatics Rose Collis PCR, Sanger sequencing Angie Reynolds gnd library preparation New Zealand Genomics Ltd at Massey University Rose Collis was the recipient of an AgResearch Core-funded Summer Studentship ( ) This work was funded through AgResearch Core (Curiosity) Funding

25 gnd as a tool for serological analysis O serogroups # known isolates Virulence Profile Source Enrichment O3/O21 2 stx1, ehxa Dairy cow Pre & Post O149:(H10) 2 stx2 Calf Pre & Post O91/O96 3 stx2 Calf Pre & Post O165:(NM) 1 stx1, stx2, eae, ehxa Calf Pre No match* 1 stx2 Calf Pre O182/O119 2 stx1, eae, ehxa Dairy cow Post O84 1 stx1, eae, ehxa Dairy cow Post * Single base pair variation compared to O104 gnd sequence from database

26 serogroup 9_112_S9counts 33_112_S33counts 57_112_S57counts 81_112_S81counts O45B O46B O123A O O90A O15B O2D id O176A O38B O153A id OND O O id O O id O id O id O174C O91E id O id O id id O156C O id id