<i>Salmonella</i> Enteritidis infection levels in spleen, liver and reproductive tract following challenge of vaccinated and naïve hens four days following intravenous challenge at varying ages through sexual development.

<p>Data are presented as median values with ranges based on five birds per group at each age and number positive for <i>Salmonella</i> out of total number challenged per group. Quantitation was not performed on pooled reproductive tract samples and are shown as <i>Salmonella</i> culture positive or negative following enrichment culture.</p>*<p>Positive only after enrichment.</p><p>ND = Not done.</p

Changes in cell populations in the chicken reproductive tract as determined by immunohistological examination.

<p>2A shows T lymphocyte populations in the infundibulum (upper oviduct), 2B in the magnum (mid oviduct) and 2Cthe ovary. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048195#pone-0048195-g002" target="_blank">Figure 2D</a> shows the changes in macrophage populations in the ovary and oviduct. Data are based on 10 fields of view for two sections of each tissue from each animal. Between five to seven birds were examined at each time point. Data are given as a mean (±SD).</p

Changes to CD4+ lymphocyte populations in the reproductive tract.

<p>At 102 days of age CD4+ populations are primarily organised into aggregates in the ovary (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048195#pone-0048195-g003" target="_blank">Figure 3A</a>) and oviduct. Birds lack lymph nodes and form more loose lymphoid structures. At 130 days (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048195#pone-0048195-g003" target="_blank">Figure 3B</a>) these structures are no longer found, with smaller populations of cells found throughout the ovary. At 165 days (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048195#pone-0048195-g003" target="_blank">Figure 3C</a>) CD4+ numbers increase slightly, but remain scattered throughout the tissue. Photomicrographs taken at a magnification of ×400.</p

Expression of Interleukin-4 (A) and Interleukin-6 (B) in the ovary.

<p>Data is expressed as 40-Ct values in ovarian tissue in comparison to expression in breast muscle tissue as a control for no expression (40-Ct = 0). RNA levels are normalised between tissues using 28S RNA expression. Data are shown as means based on expression levels of 5 birds at each time point (±SD).</p

Observed faecal and systemic <i>E. coli</i> MLST Sequence types categorised by VAG carriage.

<p>(n) <i> = </i> ST observation frequency. All faecal <i>E. coli</i> belonged to newly identified sequence types (ST) excluding ST-352. Interestingly, all ST-352 isolates harboured more than 5 VAGs with the following profiles:1) <i>astA</i>⁺, <i>irp2</i>⁺, <i>papC</i>⁺, <i>iucD</i>⁺, <i>vat</i>⁺, <i>cvi</i>⁺, <i>sitA</i>⁺ 2) <i>iss</i>⁺, <i>irp2</i>⁺, <i>papC</i>⁺, <i>iucD</i>⁺, <i>vat</i>⁺, <i>cvi</i>⁺, <i>sitA</i>⁺ 3) <i>iss</i>⁺, <i>irp2</i>⁺, <i>papC</i>⁺, <i>iucD</i>⁺, <i>vat</i>⁺, <i>cvi</i>⁺, <i>sitA</i>⁺ and they did not group with other <i>E. coli</i> in the online database. Systemic <i>E. coli</i> analysis identified 3 ST–117 and 4 ST- 2999 isolates; however ST-2998 and ST-3000 did not cluster with the other two STs in this category.</p

VAG profile diversity for all flocks.

<p><i>a</i>) 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 (<i>irp2</i>⁺, <i>papC⁺</i>, <i>va</i>t⁺,<i>cvi</i>⁺, <i>sitA</i>⁺) and 0011101010 (<i>irp2</i>⁺, <i>iucD</i>⁺, <i>papC</i>⁺, <i>va</i>t⁺, <i>sitA</i>⁺) both with 5 VAGs were the most common profile identified <i>b</i>) 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 <i>c</i>) Comparison of total number of VAGs carried by individually tested <i>E. coli</i> at t = 0 and 5. Profile 206 (0000000000) excluded from both graphs. Not all profiles were represented in all four cycles.</p

Dendrogram constructed using DICE for systemic <i>E.</i><i>coli</i>. (tolerance 5%)

<p>(minimum height >0.0%, minimum surface >0.0%)(0.0–100% coefficient) for XbaI PFGE. A dendrogram showing the strain diversity amongst systemic E. <i>coli</i> 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 (<i>irp2</i> and <i>iucD</i>) while other ST 3004 isolates have no VAGs (isolates 579 and 583).</p

Assignment of faecal and systemic <i>E. coli</i> to phylogenetic groups.

<p>216 faecal <i>E. coli</i> and 35 systemic <i>E. coli</i> from vital organs were typed using the Clermont <i>et al</i> triplex PCR and assigned to 1 of 5 phylogenetic groups. Those isolates which showed no amplification of any of the 3 targets, yet confirmed to be <i>E. coli</i> were assigned to subgroup A0. Group D was the most frequently detected phylogenetic group among faecal population, while A0 (untypable) was the most common group among systemic isolates. B2 and D have been previously associated with more pathogenic <i>E. coli</i>, however they only accounted for 28.57% of systemic isolates in this study.</p

Average percentage of pAPEC with respect to time.

<p>At weekly intervals the average percentage of potential APEC, defined by the carriage of ≥5 VAGs, from the total faecal <i>E.coli</i> population was calculated. At each time point, 160 faecal <i>E. coli</i> 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.</p

Simpson's diversity index for VAG profile diversity through time.

<p>Simpson's diversity index (D) was used to compare VAG profile diversity through time in the second flock cycles of farm 1 (F1) and farm 2 (F2). Overall, profile diversity decreases with time, with a peak at week 3.</p