Análisis de costo de los componentes utilizados

The aim of this thesis was to investigate persistence of D. nodosus at various sites on sheep and in the environment. The primary hypothesis was that there is at least one site where D. nodosus persists when there are no diseased sheep in a flock. The associations between D. nodosus presence and persistence at different sites, and levels of disease in flocks over time

were investigated using epidemiological studies of sheep and their environment with a range of molecular tools to detect, quantify and strain-type D. nodosus.

A key result of the research conducted is that D. nodosus can persist on the feet of sheep for at least 20 weeks, irrespective of the disease state of the sheep (Chapter 3). Detection of D.

nodosus on feet, in all disease states, has been reported previously (Moore et al., 2005; Calvo-

Bado et al., 2011 Witcomb, 2012) and led to the suggestion that the feet of sheep may consitute a reservoir of infection. There was, however, no evidence that D. nodosus persisted on feet. Some studies have shown that D. nodosus can be detected and quantified on the feet of sheep over time during an episode of disease (e.g Witcomb et al., 2014), but in order to obtain more relevant information on persistence, information on D. nodosus strain identity over time was needed.

We demonstrated that not only can D. nodosus be detected for 20 weeks on the feet of sheep irrespective of disease state, but one dominant strain was present on feet over time. Therefore, the MLVA tool provided valuable epidemiological information on persistence of FR that was obtained without the need to culture and will be of use in future studies of D. nodosus.

This is the first study that showed that healthy sheep can carry D. nodosus for many weeks before initiation of disease. Detection of D. nodosus on healthy feet has been reported previously (Moore et al., 2005; Calvo-Bado et al., 2011; Witcomb, 2012; Maboni et al., 2016) and is in agreement with the results obtained here, as D. nodosus was detected on the healthy feet of sheep in all 3 studies. We have shown that D. nodosus can be deteted on the

healthy feet of sheep up to 5 weeks before sheep became diseased and for 2 weeks on the foot that became diseased (study 2). Not only was D. nodosus detected, but the dominant strain identified was present throughout the 5 weeks. This key result and provides novel information on the persistence of D. nodosus. In study 3 (Spain, chapter 5), D. nodosus was detected in high loads on healthy feet when there had been no FR occurrence in the flock for at least 2 months. This supports previous suggestions that healthy feet or feet with subclinical FR act as carriers of D. nodosus. (Beveridge, 1941; Ghimire and Egerton, 1996; Moore et al., 2005; Kaler et al., 2010b).

The high detection frequency of D. nodosus in soil, grass and faecal samples in study 1 was a novel finding, overturning the existing paradigm that D. nodosus is mainly associated with the feet of sheep (Beveridge, 1941). Although indirect transmission of FR through pasture has been shown previously, suggesting that D. nodosus persists for 7-14 days (Beveridge, 1941;

Woolaston, 1993; Whittington et al., 1995), those studies did not investigate detection of D.

nodosus on pasture. Here, D. nodosus was detected in all sample types and was detected for

the first time on grass samples and in the deeper layers of soil. This indicates that D. nodosus

is able to adhere to the external environment at least for a short amount of time. This is in agreement with findings of cross-sectional studies that detected D. nodosus in soil and faecal samples collected from the pasture (Witcomb, 2012; Muzafar et al., 2015). D. nodosus growth in soil is however unlikely from a cell biology perspective, as D. nodosus has lost key biosynthetic pathways needed for survival outside the host, as illustrated by its small genome (Meyers et al., 2007). Growth, however, does not equal persistence and the facultative anaerobic nature of D. nodosus may facilitate dormancy and survival in the environment for a short amount of time, as D. nodosus is thought to remain viable under aerobic conditions for at least 10 days on pasture (Whittington et al., 1995). In vitro studies have shown that D. nodosus can remain viable at in soil microcosms between 24 and 40 days (Cederlof et al., 2013; Muzafar et al., 2016). D. nodosus may be able to invade the deeper layer of soil due to its motility, aided by sheep activity churning up the soil. Alternatively, there may be no risk of transmission to sheep, as it may perish before coming in contact with sheep again or may not be present in sufficient quantities to constitute and infectious dose.

Soil moisture plays an important role in D. nodosus survival on pasture. Smith et al. (2014) reported an increased transmission of FR when rainfall had increased 2-4 weeks previously in the UK. Graham and Egerton (1968) observed FR transmission in Australia when soil was in a saturated state for a long period of time, irrelevant of rainfall, and transmission of FR ceased when the ground dried out. In Muzafar et al. (2016) soil moisture was significantly associated with increased survival time with D. nodosus surviving longer in constantly moist soil microcosms. We did detect D. nodosus in surface and soil core samples when the ground was wet. Therefore, it could be that D. nodosus persists in damp soils for at least long enough to facilitate pathogen spread.

The number of D. nodosus positive samples on pasture was affected by dry weather in both studies. In study 1, the number of D. nodosus positive pasture samples declined as the weather became drier. In study 2, soil moisture levels declined from an average of 87% in the weeks preceding the dry weather period to an average of 50% from week 7-11. This was coupled with a reduction in the number of D. nodosus positive soil samples. Dry weather was not associated with change in survival of D. nodosus on the feet of sheep. We hypothesized that there is a positive association between D. nodosus load on the foot, in the environment

and level of disease in the flock and that environmental loads of D. nodosus correlate with disease outbreaks. We found that D. nodosus loads in the environment declined with dry weather, irrespective of the disease state of the flock, while D. nodosus load on diseased feet remained as high as 105 _{cells per swab.In contrast the host appears to become less susceptible}

to disease, as FR lesions started to heal in dry weather. No lesions were observed for 2 consecutive weeks on a sheep that had SFR from week 5 and 9. Re-appearance of SFR lesions in week 12 coincided with increasing rainfall. It is possible that dry weather promotes the healing of lesions, while D. nodosus remains viable on feet and able to cause disease. Increasing rainfall may soften the interdigital skin which facilitates invasion of D. nodosus into the interdigital skin.

It is interesting that D. nodosus was found in one soil sample in July (Study 3, Spain) when it had not rained for at least 6 weeks. Although this does not indicate that D. nodosus persisted in soil or that no sheep in the flock had FR lesions it is clear that D. nodosus can still be detected after a long period of dry and hot weather when sheep are assumed to be lesion-free. As long-term persistence of D. nodosus in dry soil is less likely, it is probable that D. nodosus was transferred to the soil from the feet of a sheep that was not sampled.

Published evidence coupled with the results from this study indicate that the climatic conditions that favour transmission of D. nodosus and survival of D. nodosus are not the same. The results of this study suggest that D. nodosus spread increases during or shortly after periods of high rainfall. D. nodosus may be able to adhere to the moist soil at least for a short amount of time after rainfall ceased and encourage further spread. Therefore, moist soil caused by preceding rainfall seems to be the key factor for transmission and also survival of D. nodosus off host. Dry weather may lead to the healing of lesions and a reduction of D.

nodosus on pasture, but the ability of D. nodosus to persist on healthy feet in periods of dry

weather shown here may explain why FR re-appears after long periods of non-transmission when it rains. Therefore, it is likely that rainfall plays a key role in initiation of disease, but not persistence of D. nodosus. For how long D. nodosus can survive on healthy feet in the absence of disease in a flock is unknown.

Some sheep with poor foot conformation (in this case thickened, misshapen or overgrown hoof horn) appear susceptible to repeated bouts of FR. These sheep are most likely to be chronically infected and such sheep were reported by the farmer and sampled in Spain. One sheep had high loads of D. nodosus, even though disease had not been observed in the flock

for at least 2 months. Sheep with poor foot conformation have also have been reported by Beveridge (1941) and Kaler et al. (2010b), as sheep that harbour D. nodosus in micro abscesses and suffer from chronic FR and may be lame for several months, although lameness might be due to lack of treatment (Kaler et al., 2012). The farmer in Spain reported that there was a high likelihood of the sheep becoming lame as soon as the weather became wet, without necessarily developing external lesions. If persistence of D. nodosus is linked to feet with a characteristic hoof conformation, farmers could improve FR control strategies targeted for treatment or removal from the flock. Control may be more challenging when carriers do not show identifying characteristics, such as poor foot conformation.

This is the first study that investigated D. nodosus in the gingival cavity with the disease status of the sheep. It was also the first to attempt to compare strains of D. nodosus between the gingival cavity and the feet of sheep. Previous studies detected D. nodosus in the gingival cavity without linking presence to disease (Bennett et al., 2009; Witcomb, 2012). The results of study 1 show that D. nodosus was significantly more likely to be present in the gingival cavity when sheep had FR. This is supported by results from study 2, where D. nodosus was detected in the gingival cavity of a sheep with SFR. Study 1 had the highest disease prevalence of all 3 studies. It was the only study were D. nodosus was detected in the gingival cavity on more than one occasion (25% of samples). In addition, strains that were present in the gingival cavity were the same strains that were the most dominant strains present on feet, as classed by MLVA. It is likely that D. nodosus re-populates the gingival cavity when disease prevalence is high. It is possible that sheep lick lesions on their feet, transferring D.

nodosus into the gingival cavity or, more likely, that D. nodosus is transferred into the mouth

from the grass.

Linked with detection of D. nodosus in the gingival cavity, we demonstrated that faecal shedding of D. nodosus occurs, although it is a rare event (Studies 2 and 3). D. nodosus had previously been detected in faeces collected from pasture (Witcomb, 2012) and from faeces on the interdigital skin (Muzafar et al., 2015). Presence of D. nodosus at these sites could be environmental contamination. Even though colonization of D. nodosus in the gingival cavity seems unlikely, it is possible that D. nodosus can passage through the digestive system of the sheep.