A longitudinal study simultaneously exploring the carriage of APEC virulence associated genes and the molecular epidemiology of faecal and systemic E. coli in commercial broiler chickens

Abstract

Colibacillosis is an economically important syndromic disease of poultry caused by extra-intestinal avian pathogenic Escherichia coli (APEC) but the pathotype remains poorly defined. Combinations of virulence-associated genes (VAGs) have aided APEC identification. The intestinal microbiota is a potential APEC reservoir. Broiler chickens are selectively bred for fast, uniform growth. Here we simultaneously investigate intestinal E. coli VAG carriage in apparently healthy birds and characterise systemic E. coli from diseased broiler chickens from the same flocks. Four flocks were sampled longitudinally from chick placement until slaughter. Phylogrouping, macro-restriction pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST) were performed on an isolate subset from one flock to investigate the population structure of faecal and systemic E. coli. Early in production, VAG carriage among chick intestinal E. coli populations was diverse (average Simpson's D value = 0.73); 24.05% of intestinal E. coli (n = 160) from 1 day old chicks were carrying ≥5 VAGs. Generalised Linear models demonstrated VAG prevalence in potential APEC populations declined with age; 1% of E. coli carrying ≥5 VAGs at slaughter and demonstrated high strain diversity. A variety of VAG profiles and high strain diversity were observed among systemic E. coli. Thirty three new MLST sequence types were identified among 50 isolates and a new sequence type representing 22.2% (ST-2999) of the systemic population was found, differing from the pre-defined pathogenic ST-117 at a single locus. For the first time, this study takes a longitudinal approach to unravelling the APEC paradigm. Our findings, supported by other studies, highlight the difficulty in defining the APEC pathotype. Here we report a high genetic diversity among systemic E. coli between and within diseased broilers, harbouring diverse VAG profiles rather than single and/or highly related pathogenic clones suggesting host susceptibility in broilers plays an important role in APEC pathogenesis.

Figure 1. Comparison of faecal and systemic E. coli VAG carriage.

Upper and lower bound 95% confidence intervals indicate statistically significant differences between VAG carriages by the two populations. Fisher's exact test indicates that irp2, papC, iucD, cvi, sitA and ibeA are significantly more associated with systemic E. coli (p<0.05) denoted in figure by *.

Figure 2. Average percentage frequency of VAGs.

(inclusive of all flocks). Average percentage frequencies of 10 VAGs were calculated and plotted against time, from t = 0 (arrival) to t  =  week 5 (depopulation). Overall, VAGs appear to decline with time, with a peak in detection at week 3 for iss, sitA and ibeA. Iron acquisition genes irp2 and iucD consistently decline with time.

Figure 3. VAG profile diversity for all flocks.

a) Shows the VAG profiles identified at t = 0 (arrival of chicks). Profiles consisting of 4 VAGs were the most diverse, with differences in iron acquisition genes being the most abundant, while profiles 0010101110 (irp2 ⁺, papC⁺, vat⁺,cvi ⁺, sitA ⁺) and 0011101010 (irp2 ⁺, iucD ⁺, papC ⁺, vat⁺, sitA ⁺) both with 5 VAGs were the most common profile identified b) Shows the VAG profiles identified at t  =  week 5. VAG profile diversity had declined over time. Most diversity was detected with the possession of 3 VAGs. No isolates carried more than 5 VAGs c) Comparison of total number of VAGs carried by individually tested E. coli at t = 0 and 5. Profile 206 (0000000000) excluded from both graphs. Not all profiles were represented in all four cycles.

Figure 4. Average percentage of pAPEC with respect to time.

At weekly intervals the average percentage of potential APEC, defined by the carriage of ≥5 VAGs, from the total faecal E.coli population was calculated. At each time point, 160 faecal E. coli were assessed. 95% upper confidence interval error bars shown. Overall, there is a general decline with time; the average detection frequency at placement of chicks (week 0) was 24.05% and only 1% by week 5.

Figure 5. Dendrogram constructed using DICE for systemic E. coli. (tolerance 5%)

(minimum height >0.0%, minimum surface >0.0%)(0.0–100% coefficient) for XbaI PFGE. A dendrogram showing the strain diversity amongst systemic E. coli harbouring APEC VAGs constructed using BioNumerics software by unweighted pair group method with Arithmetic mean. The dendrogram also shows; phylogenetic group (P) (green  =  D; red  =  A; yellow  =  B2; blue  =  B1), isolate (I), organ and age of bird at isolation (H  =  heart: K  =  kidney: Li  =  liver: Lu  =  lung; S  =  spleen), MLST sequence type (ST) and VAG profiles. The dendrogram shows the clustering of ST 117 and 2999 isolates (excluding 601) which by PFGE analysis are ∼60% different from other isolates. Several ST 3004 were identified and these potentially show the acquisition of 2 Iron acquisition genes (irp2 and iucD) while other ST 3004 isolates have no VAGs (isolates 579 and 583).