DISCUCION DE LOS RESULTADOS
CONCLUSIÓN DE LOS RESULTADOS:
Percentage feet in category (%)
Load category H-FR
H-ID H-H
increased on the healthy-feet of diseased-sheep, that changes in the bacterial load (gene copies swab-1) also changed. Changes in the F. necrophorum (rpoB) load on the healthy feet of diseased sheep were marginal in comparison to those observed for D. nodosus
(rpoD) load.
Figure 3.8. Distribution of D. nodosus and F. necrophorum population abundance on healthy-feet of healthy sheep (H-H) compared to the healthy-feet of diseased sheep (H-ID and H-FR).
Quantification data were categorised as the following: ‘U’ (undetectable): below the TDL < 103 rpoD/
rpoB copies swab-1, ‘L’ (low): 103 to < 104rpoD/rpoB copies swab-1, ‘M’ (mid): 104 to < 105rpoD/rpoB
copies swab-1, ‘Hi’ (high): 105 to < 106rpoD copies swab-1 and ‘Ht’ (highest): ! 106rpoD copies swab-1.
The D. nodosus (rpoD) and F. necrophorum (rpoB) loads were then compared, to determine when they increased (in relation to disease presentation) on firstly a (a) sheep-level analysis and secondly on a (b) foot-level analysis. D. nodosus (rpoD) and F. necrophorum (rpoB) loads were not significantly correlated in feet from healthy sheep or on feet from sheep with ID (p > 0.05) (sheep-level analysis). However, D. nodosus (rpoD) and F. necrophorum (rpoB) loads in feet from sheep with FR were significantly positively correlated (r = 0.3040; p < 0.01), with 9.0 % (r2 = 0.09) of the variance explained by the two variables (Figure 3.9.) (sheep-level analysis). D. nodosus and F. necrophorum were less frequently detected in healthy sheep and when they were detected, samples tended to be in the lower load categories - this is represented by a small tight cluster in the first scatter plot. For sheep with ID, an increase in the D. nodosus community abundance was then observed, shifting the scatter plot to the right. In sheep with FR, the scatter plot then migrated upwards, as the F. necrophorum community increased in line with the D. nodosus population.
However, when feet were analysed individually (irrespective of sheep disease status), a less obvious pattern was observed (Figure 3.9.), with D. nodosus (rpoD) and F. necrophorum (rpoB) loads correlating for healthy-feet (p < 0.01) and for feet with FR (p < 0.05). The findings from this section (Section 3.3.5.) highlight the difficulties associated with analysing such a complex data set; consisting of longitudinal time points, stages of disease and quantification data for two pathogens. The data therefore required additional analysis, in the form of a statistical model, in order to find significant patterns associated with disease initiation and progression and F. necrophorum (rpoB) and D. nodosus (rpoD) load.
H Feet Feet with ID Feet with FR log 10 rpoB copies swab -1
log10rpoD copies swab-1
(U)
(U) (U) (U)
FOOT-LEVEL ANALYSIS 0 2 4 6 8 0 2 4 6 8 0 2 4 6 8 0 2 4 6 8 R2 = 0.0035 ns R2 = 0.2380 p < 0.05 R2 = 0.0993 p < 0.01 SHEEP-LEVEL ANALYSIS 0 2 4 6 8 0 2 4 6 8 0 2 4 6 8 0 2 4 6 8 log 10 rpoB copies swab -1
log10rpoD copies swab-1
H Sheep
(U)
Sheep with ID Sheep with FR
(U) (U) (U) R2 = 0.0467 ns R2 = 0.0206 ns R2 = 0.0924 p < 0.01
Figure 3.9. Correlation analysis between D. nodosus (rpoD) and F. necrophorum (rpoB) load in healthy sheep (H), sheep with ID and sheep with FR (sheep-level analysis) and in healthy feet (H), feet with ID and feet with FR (foot-level analysis). A significant positive correlation was observed between D. nodosus and F. necrophorum load in sheep with FR (p > 0.01) (sheep-level analysis). A less obvious pattern was observed for the foot-level analysis; D. nodosus(rpoD) and F. necrophorum (rpoB) loads appeared to correlate for healthy-feet (p < 0.01) and for feet with FR (p < 0.05). Quantification data were +1 log10 transformed and analysed using Pearson’s correlation test (two-tailed) (GraphPad Prism software). Undetectable (U) values were coded as +1 and then log10 transformed, so that they were represented on the scatter plots (as zero).
3.3.6. Construction of a multinomial regression model to elucidate the temporal patterns of D. nodosus (rpoD) and F. necrophorum (rpoB) load associated with the development and presentation of ID and FR in sheep.
The quantitative data obtained from the longitudinal study were also analysed using an unordered multinomial mixed effects model for repeated observations and samples of feet clustered within ewes using MLwiN 2.2.1 as described in Section 2.20. Multiple regression models can incorporate many explanatory variables, in this case the presence of D. nodosus and F. necrophorum split into load categories one week prior to disease (ID and FR) development and week of disease (ID and FR) onset. Univariate analysis would assume that the response variable (ID and FR) is influenced only by one factor. By using multivariable regression one can take into consideration multiple predictive factors, providing more flexibility and accuracy. Comparison of multivariable and univariate analysis are shown in Table 3.6.
For the multivariable analysis - after accounting for a lack of independence between sheep and sampling events and variability in incidence of disease by week, detection of
D. nodosus (rpoD) at the low (OR: 2.363, 95 % CIs: 1.011-5.217) and mid (OR: 2.547, 95 % CIs: 1.048-6.190) ranges was significantly more likely in feet the week before the onset of ID, however there was then a significant association between the ‘high’ (OR: 3.391, 95 % CIs: 1.401-8.207) and ‘highest’ (OR: 5.613, 95 % CIs: 2.152-14.635) ranges the week of ID onset (Table 3.6/Figure 3.10.). Indicating an increase in D. nodosus (rpoD) load during the progression from a healthy foot to a foot presenting with ID within one week. In addition, a foot was more likely to develop FR when
D. nodosus (rpoD) was detectable one week prior to the development of FR and the strength of this association increased when D. nodosus (rpoD) load was at its highest (OR: 27.385, 95 % CIs: 3.980-188.414) (i.e. was dose-dependent). Interestingly, no significant association was observed between D. nodosus (rpoD) load and FR presentation (the week of disease onset) (Table 3.6/Figure 3.10.). In contrast, F. necrophorum (rpoB) load was not significantly associated with ID or FR, the week prior to disease onset or the week of ID onset (Table 3.6/Figure 3.10.). However, an association between FR and F. necrophorum (rpoB) was observed, but only after FR had developed (week of disease) with a high (OR: 5.518, 95 % CIs: 1.330-22.896) F. necrophorum load being significantly associated with FR presentation. Which is consistent with the descriptive analyses (Section 3.3.5.).
The univariate results (Table 3.6.) demonstrate that by only examining single predictors most associations are found to be significant. However, by examining the effects of D. nodosus and F. necrophorum load simultaneously in the development of ID and FR, the effect of D. nodosus in the progression of a healthy foot to one presenting with ID and in the development of FR is considered significant, whereas F. necrophorum load (‘high’ category) is now only significant after lesion (FR) development.
Univariate
Univariate MultivariableMultivariable Explanatory
variablesa Odds ratios with ID
95 % CIs (lower, upper)
FR Odds ratios with 95 % CIs (lower,
upper)
ID
Odds ratios with 95 % CIs (lower,
upper)
FR
Odds ratios with 95 % CIs (lower, upper) Week prior to onset Dn undetectable - - - - Dn low 2.389 (1.063, 5.368) 1.024 (0.220, 4.762) 2.363 (1.011, 5.217) 2.255 (0.383, 13.287) Dn mid 3.019 (1.344, 6.783) 2.472 (0.665, 9.191) 2.547 (1.048, 6.190) 8.248 (1.767, 38.496) Dn high 4.108 (1.783, 9.468) 1.680 (0.359, 7.858) 1.813 (0.693, 4.746) 3.515 (0.545, 22.668) Dn highest 3.892 (1.435, 10.555) 5.835 (1.440, 23.652) 2.630 (0.815, 8.492) 27.385 (3.980, 188.414) Fn undetectable - - - - Fn low 3.501 (1.413, 8.675) 1.423 (0.154, 13.257) 0.745 (0.296, 1.870) 0.302 (0.036, 2.546) Fn mid 1.844 (0.879, 3.869) 1.210 (0.342, 4.285) 0.726 (0.375, 1.406) 0.526 (0.176, 1.577) Fn high 4.674 (1.782, 12.260) 5.842 (1.450, 23.537) 1.326 (0.502, 3.498) 1.272 (0.294, 5.513) Week of onset Dn undetectable - - - - Dn low 1.441 (0.659, 3.149) 0.678 (0.191, 2.404) 1.283 (0.560, 2.939) 2.113 (0.558, 7.995) Dn mid 2.143 (1.002, 4.584) 0.816 (0.230, 2.901) 1.968 (0.857, 4.518) 0.407 (0.107, 1.557) Dn high 4.166 (1.932, 8.983) 1.333 (0.373, 4.768) 3.391 (1.401, 8.207) 0.489 (0.112, 2.134) Dn highest 6.773 (2.997, 15.301) 2.945 (0.880, 9.849) 5.613 (2.152, 14.635) 0.711 (0.158, 3.245) Fn undetectable - - - - Fn low 2.121 (1.077, 4.179) 1.107 (0.216, 5.678) 1.770 (0.807, 3.884) 1.564 (0.359, 6.814) Fn mid 0.729 (0.418, 1.272) 1.412 (0.498, 4.006) 0.907 (0.483, 1.701) 2.228 (0.749, 6.624) Fn high 1.214 (0.530, 2.782) 4.371 (1.315, 14.534) 0.955 (0.374, 2.437) 5.518 (1.330, 22.896) Week 0.445 (0.353, 0.561) 0.107 (0.071, 0.161) 1.619 (1.240, 2.114) 2.018 (1.229, 3.313)
Table 3.6. Output from full multinomial regression model (univariate and multivariable results) for the effect of D. nodosus (Dn) (rpoD) and F. necrophorum (Fn) (rpoB) load on the development and presentation of ID and FR. The baselines for each explanatory variable were Dn undetectable (U) and Fn undetectable (U). Quantification data were categorised as the following: ‘U’ (undetectable): < 103
copies swab-1, low: 103 to < 104 copies swab-1, mid: 104 to < 105 copies swab-1, high: 105 to < 106 copies
swab-1 and highest: ! 106rpoD copies swab-1. The high category for F. necrophorum data was ! 105rpoB