Principios Cardinales de Atalaya

The present study revealed an overall prevalence of T. parva carrier state to be 34.5%. This value was close to a 37.1% prevalence found by Kazungu et al. (2015). While this study sampled cattle from Simanjiro and Manyara districts in the Maasai steppe and farms in eastern Tanzania the study by Kazungu et al. (2015) sampled cattle from Simanjiro district of the Maasai steppe and farms in Mwanza, Lake Zone of Tanzania. Hence the difference

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in T. parva prevalence between the two studies is likely due to different tick control regimes in the different areas. The prevalence of T. parva established in cattle in this study was higher than a 31.6% prevalence found by Kimaro, Mor, Gwakisa and Toribio (2017) which was done in Monduli district in the Maasai steppe ecosystem during the dry season. These differences in prevalence may be explained by the ecological and other associated conditions which may favor ticks in different areas where cattle were sampled. The study done by Kimaro et al. (2017) sampled cattle during the dry season which is characterized with low tick intensity compared to the wet season during which the present study was conducted. The prevalence values reported in these studies point to a high possibility that most of the animals sampled were in a T. parva carrier state, which is a common phenomenon following natural infection or ITM in cattle (Oura et al., 2007; Oura et al., 2011).

The cattle groups in this study were sampled from divergent ecological conditions and management regimes for controlling ticks and tick-borne diseases. It was of interest therefore to compare the cattle groups not only in terms of prevalence of T. parva but also how the prevalence varies within and between groups, when the cattle are categorized with respect to whether they were ECF vaccinated or not, longevity of T. parva carrier state following ECF vaccination and proximity of cattle grazing areas to wildlife interface areas. Overall a higher T. parva prevalence was clearly shown among cattle that were ECF- vaccinated (43%) compared to unvaccinated cattle (13.4%). This finding is in full agreement with the expected norm that ECF vaccination using the live trivalent Muguga cocktail increases the carrier state of T. parva among cattle. Likewise, a higher T. parva prevalence was found among cattle which grazed in close proximity to the wildlife interface. The higher prevalence (38%) of T. parva infection among cattle grazing close to the wildlife interface areas may be explained by the fact cattle that share grazing areas with wildlife reservoirs are likely to be under higher exposure to disease vectors and hence higher T. parva positivity.

In order to get a better understanding of longevity or persistence of carrier state following ITM under prevailing conditions in the study areas, this study classified all ECF vaccinated cattle based on their T. parva positivity. All the 43% (103/239) T. parva positive ECF-vaccinated cattle were further clustered into categories of longevity (duration) since ECF vaccination. The data showed that carrier state was detected in cattle

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vaccinated as far as 11 years ago. Comparison of categories of longevity since vaccination revealed that highest frequency of carriers was at 12 months post vaccination these results further confirm previous reports by Oura et al. (2007) who detected the carrier state of T. parva only up to 4 years. The results reported here have taken advantage of deployment of ITM in the Maasai steppe for at least two decades, and therefore the availability of older cattle vaccinated up to 14 years earlier has provided a useful resource to investigate ITM outcomes in ECF endemic areas.

An interesting question arising from this study was whether the carrier state can be differentiated between ECF vaccinated cattle and those that may have recovered from natural ECF infection. Three VNTR markers, which are constituent components of the Muguga cocktail, were used to monitor carrier state in T. parva positive ECF unvaccinated (n=13) cattle. It emerged that 76.9% of this group carried the Muguga vaccine markers (MS 7 and ms 5) indicating that vaccine strains are transmissible to the unvaccinated cattle. Since majority of these cattle belonged to one farm (Leila farm) it may likely be that this is a result of management conditions supporting the transmission of vaccine strains from vaccinated to unvaccinated cattle. This study showed that two of the three vaccine markers (MS 7 and ms 5) were detected in unvaccinated cattle. Although the ms 2 marker was not detected it is uncertain whether this was due to small sample size used in the present work or rather, this study further supports previous findings by Oura et al. (2 0 0 7 ) where he showed evidence of transmission of some and not all Muguga vaccine components.

The implication of long-term deployment of the ITM, as is the case in northern Tanzania, has not been investigated. Prior to this study, it was hypothesized that long term application of ITM may influence parasite biology, alter genetic diversity of local strains and hence impact on disease dynamics in endemic areas. Although ITM has been employed to protect cattle against ECF in the Maasai herds since 1990s (Di Giulio et al., 2009) no study has preceded this one to investigate T.

parva genetic diversity in Tanzania. Genotyping of the T. parva positive samples using the three

VNTR markers allowed to unravel the genetic diversity of T. parva in cattle from different locations. The extent of polymorphism varied between the three markers, whereas ms 5 was most polymorphic and MS 7 was least.

Higher genetic diversity was observed among ECF vaccinated cattle compared to unvaccinated cattle. Thus, the 24 alleles across the three markers were all detected with varying frequencies among vaccinated cattle, but only 4 of these were found in

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unvaccinated cattle. It is probable that vaccinated cattle would harbor more alleles due to recombination of the local strains with vaccine strains; another explanation for this finding may be due to the ability of the carrier cattle acquiring infection with additional parasite genotypes following tick challenge, thus resulting in carriage of mixed genotypes. This can be used to support the finding that the Manyara ranch group, found at the ecological epicenter of the wildlife interface within the Maasai steppe had the highest mean number of different alleles, effective alleles, expected heterozygosity and private alleles.

While majority of the alleles were shared by individuals from different groups, this study found seven private alleles, each of which was found only in one or the other group. Interestingly all of the private alleles were detected only in vaccinated cattle grazing close to the wildlife interface. The actual mechanisms supporting existence of private alleles are not fully understood but these findings may be ascribed to the notion that the Muguga vaccine is tri-valent, with a multitude of parasite genotypes (Oura et al., 2007; Patel et al., 2011).

In order to gain insight in the population diversity of T. parva circulating in the different cattle groups the principle component analysis (PCA) was used. Results revealed different clustering patterns, with most of the alleles clustering together throughout all the four quadrants. The PCA findings strongly suggest the T. parva parasites homogeneity among the cattle groups and the absence of a clear association between population genetic structure and the geographical origin of the isolates. Furthermore, analysis of molecular variance revealed higher genetic variations within individual isolates (97%) compared to within T. parva populations (3%). One possible explanation to this finding is the occurrence of a high rate of crossing between different T. parva isolates and recombination within the parasite population hence the lower diversity within the T. parva populations. This was somehow surprising considering that cattle groups sampled from Tanga (eastern Tanzania) were expected to carry distinct satellite alleles, separate from thosefound in cattle from the Maasai steppe (northern Tanzania). It is possible that uncontrolled cattle movements between the different regions in Tanzania may have contributed to the similarity of the parasite genotypes.

The use of ITM in Tanzania has been increasing gradually since 1998 (Di Giulio et al., 2009; Martins et al., 2010). This served as one of the motives for the present study in order to understand implications of the long term deployment of ITM for over 20

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years in the Maasai areas, Thus the study has brought forth clear findings showing that the carrier state induced by Muguga cocktail vaccine is effective and it induces a long lasting immunity detectable up to 11 years post vaccination something which hitherto has not been reported. Furthermore, results of this study have allowed to deduce the questionable viewpoint on the spreading of the vaccine strains to the unvaccinated cattle via tick vectors. In this study, no ticks were investigated, notwithstanding it was clearly demonstrated that two of the three vaccine markers were detected in several co-grazing unvaccinated cattle. These findings corroborate previous reports by Olds et al. (2018).

A significant finding emanating from this study is that vaccination against ECF has an influence on the diversity of T. parva parasites, whereby greater number of alleles were shown in the vaccinated cattle compared to the unvaccinated cattle. It was speculated that the enhancement of diversity is a direct outcome of the live vaccination process in the field, whereby the vaccine strains may potentially recombine with local strains to generate more genotypes. It may be speculated that wider T. parva diversity plays a significant role to restrict breakthrough infections in the vaccinated cattle, as was observed during the conduct of this study, whereby no clinical ECF cases were encountered among sampled cattle. The study took into consideration separate ecological and geographical locations from where the samples were collected. Interestingly, analysis of T. parva populations revealed that geographical separation did not necessarily imply differences in the genetic structure of T. parva populations.

Summing up, majority of the cattle investigated in this study were sampled from wildlife interface areas. Such areas support constant interaction between cattle, wildlife reservoirs, tick vectors and the parasites. Ecological pressure in such an interface presumably drive the establishment of a carrier state in cattle differently as it would happen in cattle populations grazed far from wildlife. Therefore, the role of the wildlife interface on the diversity of T. parva may not be negated, as highest parasite diversity shown in this study was among ECF vaccinated cattle found in close proximity to wildlife interface.

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CHAPTER FIVE

CONCLUSION AND RECOMMENDATIONS