Orientador: Gilson Pereira de Oliveira / Coorientador: Alvimar José da Costa / Banca: Marcos Rogério André / Banca: Cláudio Alessandro Massamitsu Sakamoto / Resumo: Ao longo das últimas décadas o controle de Rhipicephalus (Boophilus) microplus tem sido dificultado pelo desenvolvimento da resistência à maioria dos grupos químicos utilizados. Algumas moléculas tem se apresentado como alternativas promissoras, mas mesmo assim, apresentam redução de eficácia com o passar do tempo. As lactonas macrocíclicas, moléculas com atividade endectocida, surgiram como uma dessas opções, mas relatos sobre a resistência, especialmente à ivermectina, se tornam cada vez mais frequentes. O presente estudo objetivou determinar a redução de eficácia da ivermectina contra o R. (B.) microplus, e o consequente surgimento da resistência à esse princípio ativo, por meio de resultados encontrados em várias propriedades rurais, frente às diferentes cepas presentes. Foram realizados testes com bovinos naturalmente infestados da região Sudeste do Brasil, utilizando diferentes concentrações de ivermectina (200 μg/kg, 500 μg/kg e 630 μg/kg). Estes experimentos basearam-se na contagem de partenóginas, entre 4,5 e 8 mm de diâmetro, presentes no lado esquerdo dos bovinos. Das doze propriedades avaliadas, três com a utilização de ivermectina 500 μg/kg (0,5% pour-on), três onde foi testada a ivermectina 200 μg/kg (1% injetável) e seis onde avaliou-se a ivermectina 630 μg/kg (3,15% injetável), apenas uma apresentou valores de eficácia média, entre os dias 7 e 14 pós-tratamento, superior à 90%, sendo classificada como sensível. Nenhum dos demais experimentos atingiu este valor, permitindo a classificação de onze dentre as doze cepas (91,67%) avaliadas, como resistentes. Além disso, o presente estudo confirma a eficiência da avaliação in vivo para o diagnóstico da resistência, assim como reforça a necessidade de maior atenção a esse fenòmeno, amplamente disseminado nessa região do Brasil / Abstract: Throughout the last decades, the control of Rhipicephalus (Boophilus) microplus has become more difficult due to the development of resistance to most of the products used for its control. Some molecules have been presented as promising alternatives, but nonetheless, their efficacy has shown reduction as time passes. The macrocyclic lactones, molecules with endectocidal activity, appeared as one of these options, but resistance to them, especially to ivermectin, are commonly reported in literature. The present study aimed to determine the decrease in acaricidal efficacy of ivermectin against Rhipicephalus (Boophilus) microplus, and the consequent development of resistance to this active component, by means of several results found in several rural properties, with different tick strains present. Tests were conducted in naturally infested animals originated from farms on the Southeast region of Brazil, using different concentrations of ivermectin (200 μg/kg, 500 μg/kg and 630 μg/kg) applied in different administration methods (pour on and injectable). From all twelve properties evaluated, being three with the administration of 500 μg/kg ivermectin (0.5% pouron), three were 200 μg/kg ivermectin (1% injectable) was tested and six were 630 μg/kg ivermectin (3.15% injectable) was evaluated, only one presented mean efficacy values, between days 7 and 14 post-treatment, superior to 90%, being classified as sensible. None of the other experiments reached or surpassed this index, allowing the classification of eleven amongst twelve (91.67%) evaluated R. (B.) microplus strains, as resistant. Besides, the present study confirms the efficiency of in vivo evaluation for the diagnosis of resistance, as well as reinforces the need for a greater attention to this problem, widely spread in this region of Brazil / Mestre

The purpose of this study was to determine the prevalence of Lyme Disease (LD) bacteria in adult Ixodes pacificus ticks collected from the mountains of San Bernardino County in Southern California. Seven hundred fifty four I. pacificus adults were collected from the Pacific Crest Trail and adjacent areas. The Polymerase Chain Reaction (PCR) was used to screen ticks for Borrelia burgdorferi infection by targeting two different DNA loci. Oligonucleotide primers targeting both the ospA and fla genes were used in the assay. Ticks were processed in pools of three, and genomic DNA from the ticks was extracted with a commercial mini-kit utilizing silica matrix spin-columns. All ticks tested negative for B. burgdorferi infection regardless of primer pair used. In addition, ticks were negative following examination by dark-field microscopy. This study confirms previous reports that the prevalence of LD in Southern California is quite low.

Ticks are of global interest as the pathogens they spread can cause diseases that are of importance to both human health and economies. In Scotland, the most populous tick species is the sheep tick Ixodes ricinus, which is the vector of pathogens causing diseases such as Lyme borreliosis and Louping-ill. Recently, both the density and spread of I. ricinus ticks have grown across much of Europe, including Scotland, increasing disease risk. Due to the nature of the tick lifecycle they are particularly dependent on environmental factors, including temperature and habitat type. Because of this, the recent increase in tick-borne disease risk is believed to be linked to climate change. Many mathematical models have been used to explore the interactions between ticks and factors within their environments; this thesis begins by presenting a thorough review of previous modelling of tick and tick-borne pathogen dynamics, identifying current knowledge gaps. The main body of this thesis introduces an original mathematical modelling framework with the aim to further our understanding of the impact of climate change on tick-borne disease risk. This modelling framework takes into account how key environmental factors influence the I. ricinus lifecycle, and is used to create predictions of how I. ricinus density and disease risk will change across Scotland under future climate warming scenarios. These predictions are mapped using Geographical Information System software to give a clear spatial representation of the model predictions. It was found that as temperatures increase, so to do I. ricinus densities, as well as Louping-ill and Lyme borreliosis risk. These results give a strong indication of the disease risk implications of any changes to the Scottish environment, and so have the potential to inform policy-making. Additionally, the models identify areas of possible future research.

Text in English / Corridor disease is an economically important tick-borne disease of cattle in southern Africa. The disease is caused by Theileria parva and transmitted by the vectors, Rhipicephalus appendiculatus and R. zambeziensis. There is currently no vaccine to protect cattle against T. parva that is permitted in South Africa. To develop recombinant anti-tick vaccines against Corridor disease, comprehensive databases of genes expressed in the tick’s salivary glands are required. Therefore, in Chapters 2 and 3, mRNA from the salivary glands of R. appendiculatus and R. zambeziensis was sequenced and assembled using next generation sequencing technologies. Respectively, 12 761 and 13 584 non-redundant protein sequences were predicted from the sialotranscriptomes of R. appendiculatus and R. zambeziensis and uploaded to public sequence domains. This greatly expanded the number of sequences available for the two vectors, which will be invaluable resources for the selection of vaccine candidates in future. Further, in Chapter 3, differential gene expression analysis in R. zambeziensis revealed dynamic expression of secretory protein transcripts during feeding, suggestive of stringent transcriptional regulation of these proteins. Knowledge of these intricate expression profiles will further assist vaccine development in future. In Chapter 4, comparative sialotranscriptomic analyses were performed between R. appendiculatus and R. zambeziensis. The ticks have previously shown varying vector competence for T. parva and this chapter presents the search for correlates of this variance. Phylogenetic analyses were performed using these and other publically available tick transcriptomes, which indicated that R. appendiculatus and R. zambeziensis are closely related but distinct species. However, significant expression differences were observed between the two ticks, specifically of genes involved in tick immunity or pathogen transmission, signifying potential bioinformatic signatures of vector competence. Furthermore, nearly four thousand putative long non-coding RNAs (lncRNAs) were predicted in each of the two ticks. A large number of these showed differential expression and suggested a potential transcriptional regulatory function of lncRNA in tick blood feeding. LncRNAs are completely unexplored in ticks. Finally, in Chapter 5, concluding remarks are given on the potential impact the R. appendiculatus and R. zambeziensis sialotranscriptomes may have on future vaccine developments and some future research endeavours are discussed. / Life and Consumer Sciences / Ph. D. (Life Sciences)

Indiana University-Purdue University Indianapolis (IUPUI) / The Lyme disease agent, Borrelia burgdorferi, has a complex system that allows it to thrive in the harsh and distinct environments of its tick vector and mammalian host. Although it has been known for some time that the Borrelia oxidative stress regulator protein (BosR) plays a necessary role in mammalian infectivity and functions as a transcriptional regulator of alternative sigma factor RpoS, very little is known about its mechanism of action, other than the suggestion that BosR activates rpoS transcription by binding to certain upstream regions of the gene. In our studies, we performed protein degradation assays and luciferase reporter assays for further understanding of BosR function. Our preliminary findings suggest that BosR is post-transcriptionally regulated by an unknown protease and may not need to bind to any rpoS upstream regions in order to activate transcription. We also describe the construction of luciferase reporter systems that will shed light on BosR’s mechanism of action. We postulate the provocative possibility that unlike its homologs Fur and PerR in other bacterial systems, BosR may not utilize a DNA-binding mechanism in order to fulfill its role as a transcriptional regulator to modulate virulence gene expression.