Ce travail s’articule sur trois axes ; le premier est une contribution à l'étude du répertoire des bactéries associées aux arthropodes vecteurs (tique et puces) en Afrique du nord (Algérie) et en Afrique Sub-saharienne (Bénin, Tanzanie et République Démocratique du Congo). Nous avons pu ainsi détecter par biologie moléculaire (qPCRs, PCR standard et séquençage) et pour la première fois au Bénin, Rickettsia typhi (l'agent du typhus murin), et Bartonella sp dans des puces collectées sur des rongeurs à Cotonou. Dans ce travail, nous avons également détecté Yersinia pestis, l'agent de la peste et R. felis (responsable de la fièvre boutonneuse) dans des puces de la RD du Congo. En Tanzanie, nous avons mis en évidence la présence de R. felis et R. typhi dans des puces de rongeurs. En Algérie, nous avons décrit pour la première fois la présence d'agent de borréliose de Lyme (Borrelia garinii) dans les tiques. Nous avons confirmé la présence de R. massiliae, R. monacensis R. aeschlimannii, R. slovaca et R. felis et nous avons également détecté pour la première fois en Algérie, Bartonella tamiae, une bactérie dont la pathogénicité est peu connue et Coxiella burnetii, l'agent de la fièvre Q.Dans la deuxième partie de notre travail, nous nous sommes intéressés à l’évaluation des compétences vectorielles des puces de chat (Ctenocephalides felis) et punaises de lit (Cimex lectularius) dans la transmission de l’agent de la fièvre des tranchées (Bartonella quintana) dont le vecteur connu est le pou de corps. Trois approches ont été utilisées : la qPCR, la culture et l’immunohistochimie. / This work focuses on three areas; the first is a contribution to the study of the repertoire of bacteria associated with arthropod vectors (tick and flea) in North Africa (Algeria) and in Sub-Saharan Africa (Benin, Tanzania and the Democratic Republic of Congo). We could thus detected by molecular tools (qPCRs, standard PCR and sequencing) and for the first time in Benin, Rickettsia typhi (the agent of murine typhus) and Bartonella sp in fleas collected from rodents in Cotonou. In this work, we have also associated the agent of plague (Yersinia pestis), and for the first time in fleas of DR of Congo, and we detected also R. felis (the causative agent of spotted fever). In Tanzania, we have highlighted the presence of R. typhi and R. felis fleas on rodents. In Algeria, we described for the first time the presence of Lyme disease agent (Borrelia garinii) in hard ticks. We confirmed the presence of R. massiliae, R. monacensis, R. aeschlimannii, R. slovaca and R. felis, we also detected for the first time Bartonella tamiae and Coxiella burnetii associated with bat ticks in Algeria.Regarding the second part we was interested in the evaluation of vector competence of cat fleas (Ctenocephalides felis) and bed bugs (Cimex lectularius) in the transmission of trench fever agent (Bartonella quintana) that is known to be transmitted by lice. Three approaches have been tested; qPCR, culture and immunohistochemistry.

Philosophiae Doctor - PhD / Tortoises appear to be successful in arid ecosystems, where they depend on primary production for their predominantly herbivorous diets. The low primary production of arid regions is exacerbated by priodic droughts, so that iteroparous species such as chelonians require mechanisms to overcome resource shortages. The smallest of all tortoises, Homopus signatus signatus, occurs in a dry winter rainfall area in northwestern South Africa that is threatened with aridification due to regional climate change. The overall aim of this study is to understand the ecology of H. s. signatus, particularly in reference to the importance of rainfall.

"Ticks have already been studied in our country and abroad. Progress in the field of taxonomy has probably been the most rapid and has culminated in a large number of papers and excellent monographs, but much more remains to be done. To produce an overall picture of the factors influencing the behaviour of the tick under natural conditions, studies have to be conducted on tick ecology and experimental physiology. We still have a lack of knowledge of host specificity, the selection of feeding sites on hosts and the nature of resistance to attacks by ticks. Because of the varied habits of different species of ticks, the first essential prerequisite is an adequate knowledge of their biology." (Jansen 1969). The above words sum up the position of research on ticks in southern Africa at the present time.

Casos fatais de Febre Maculosa são registrados no estado do Rio de Janeiro. Avaliou-se o status epidemiológico de riquétsias Grupo Febre Maculosa em cães e ectoparasitas de seis municípios usando métodos imunológico (Reação de Imunofluorescência Indireta) e moleculares (Reação em Cadeia pela Polimerase convencional e em Tempo Real e Análise de Sequências). Avaliou-se a funcionalidade de iniciadores para a região VNTR (Número Variável de Repetições Sequenciais) de Rickettsia rickettsii, para genotipagem de amostras brasileiras. Registraram-se cães sorologicamente positivos em todos os municípios e gene riquetsial (gltA) foi detectado em seus soros, indicando sua importância na epidemiologia de riquétsias na região Metropolitana. R. rickettsii foi diagnosticada em Amblyomma cajennense sugerindo o envolvimento deste no surto com casos fatais do município de Petrópolis. Os iniciadores VNTRB mostraram-se limitados para genotipagem das cepas de R. rickettsii. O estudo evidenciou a necessidade de um sistema de vigilância ambiental contínuo para prevenção de casos fatais. / Spotted Fever fatal cases are reported in Rio de Janeiro state. We evaluated the epidemiological status of Spotted Fever Group Rickettsiae in dogs and ectoparasites of six municipalities using immunological methods (Indirect Immunofluorescence Assay) and molecular (Conventional and Real-Time Polymerase Chain Reaction and Sequence Analysis). We evaluated the functionality of primers for the VNTR region (Variable Number Tandem Repeat) of Rickettsia rickettsii, for brazilian samples genotyping. Dogs serologically positive were registered in all municipalities and riquetsial gene (gltA) was detected in their serum, indicating its importance in rickettsiae epidemiology in Metropolitan region. R. rickettsii was diagnosed in Amblyomma cajennense suggesting an involvement of this species in an outbreak with fatal cases in Petrópolis city. Primers VNTRB proved limited for genotyping R. rickettsii strains. This study highlighted the need for a continuous environmental monitoring system to prevent fatal cases.

Rickettsia rickettsii é o agente etiológico da Febre Maculosa das Montanhas Rochosas, que no Brasil é transmitida pelos carrapatos Amblyomma cajennense e A. aureolatum. Para elucidar os mecanismos de virulência sobre seus vetores, construímos bibliotecas subtrativas utilizando RNA de A. aureolatum infectados ou não com o patógeno. Com a análise bioinformática, foram obtidas 56 sequências únicas com expressão induzida e 12 com expressão reprimida pela infecção. Após a validação dos dados por RT-qPCR 3 genes foram caracterizados por RNAi: uma hebraeína, uma proteína dissulfeto isomerase (PDI) e uma proteína com domínio Kunitz-type. Um maior número de carrapatos adquiriu R. rickettsii quando a expressão gênica da hebraeína e da PDI foi silenciada, sugerindo que elas participam na defesa do carrapato contra a infecção. Nenhum efeito foi observado sobre a transmissão da bactéria para o hospedeiro ou sobre o fitness de carrapatos nos três genes analisados. O presente estudo apontou genes importantes que possibilitam uma melhor compreensão da relação carrapato-riquétsia. / Rickettsia rickettsii is the etiological agent of Rocky Mountain Spotted Fever and, in Brazil, it is transmitted by Amblyomma cajennense and A. aureolatum. To elucidate mechanisms of virulence to its vectors, we construct cDNA libraries with RNA of ticks A. aureolatum infected or not with this pathogen. After bioinformatic analysis, 56 unique sequences were obtained representing up-regulated genes and 12 down-regulated by infection. After data validation by RT- qPCR, 3 genes were characterizated by RNAi: a hebraein, a protein disulfide isomerase (PDI), and a protein with Kunitz-type domain. A higher number of ticks acquired R. rickettsii when the gene expression of hebraein and PDI was silenced, suggesting that both proteins participate in the defense of the tick against infection. No effect on the transmission of the bacterium to the host or on the fitness of ticks was observed after knockdown of the 3 analyzed genes. Data obtained by the present study pointed out important genes that provide information to better understand of the tick-rickettsia relationship.

Orientador: Ricardo Velludo Gomes de Soutello / Resumo: O objetivo do trabalho foi caracterizar um rebanho de 72 novilhas ½ Angus x ½ Nelore, identificando os animais resistentes, resilientes esuscetíveis a endo e ectoparasitas, correlacionando a metilação global do DNA desses animais e o grau de parasitismo, que foi avaliado a partir da contagem de ovos por grama de fezes (OPG), moscas-dos-chifres e carrapatos. Essas contagens foram submetidas a análise de variância (ANOVA) para agrupamento em resistentes, resilientes e suscetíveis pelo teste Scott-Knott (p<0,05) noprograma SISVAR e agrupamento hierárquico no programa GENEs. O conteúdo de metilação foi analisado a partir do DNA extraído do sangue das novilhas, por meio dekit de metilação, e a quantificação feita por leitora de microplacas. Os valores absolutos médios de absorbância dos três grupos foram submetidos à ANOVA e análises pelo Tukey (p<0,05). Observou-se 36,11% de animais resistentes, 52,78% resilientese 11,11% suscetíveisa infecção por helmintos gastrintestinais, com contagens de 86, 392 e 1087 OPG, e 0,238, 0,225 e 0,197 do DNA metilado, respectivamente. Para a infestação por carrapatos, 26,39% dos animais foram classificados como resistentes, 59,72% resilientes e 13,98% suscetíveis, com contagensde 3, 8 e 19 carrapatos e 0,295, 0,216 e 0,151 do DNA metilado, respectivamente. E para a infestação por mosca-dos-chifres observou-se 54,17% de novilhas resistentes e 45,83% suscetíveis, com contagensde 17 e 32 moscas e 0,245 e 0,205 do DNA metilado, respectivamente. Na cla... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The objective of the work was to characterize a herd of 72 heifers ½ Angus x ½ Nellore, identifying the resistant, resilient and susceptible animals to endo and ectoparasites, correlating the overall DNA methylation of these animals and the degree of parasitism, which was evaluated from the egg count per gram of feces, horn flies and ticks.These counts were submitted to analysis of variance ANOVA for grouping in resistant, resilient and susceptible by the Scott-Knott test (p <0.05) in SISVAR and hierarchical grouping in the GENEs program. The methylation content was analyzed from the DNA extracted from the blood of the heifers, using a methylation kit and quantification by a microplate reader. The mean absolute values of absorbance of the three groups were submitted to ANOVA and analysis by Tukey (p <0.05). There were 36.11% of resistant animals, 52.78% resilient and 11.11% susceptible to gastrointestinal helminth infection, with counts of 86, 392 and 1087 OPG, and 0.238, 0.225 and 0.197 of methylated DNA, respectively. For tick infestation, 26.39% of the animals were classified as resistant, 59.72% resilient and 13.98% susceptible, with counts of 3, 8 and 19 ticks and 0.295, 0.216 and 0.151 of methylated DNA, respectively. And for the horn fly infestation, 54.17% of resistant and 45.83% susceptible heifers were observed, with counts of 17 and 32 flies and 0.245 and 0.205 of methylated DNA, respectively. In the second classification, 33.33% of the animals were classified as r... (Complete abstract click electronic access below) / Mestre

Since ticks are classified as hematophagous ectoparasites, the primary feeding event involves a bloodmeal on a vertebrate host. Such activities facilitate the ingestion of microorganisms which may be detrimental to the survival of a tick. It is observed, however, that ticks are able to survive such invasion by microorganisms and in several cases, facilitate the transmission of pathogens, while themselves remaining unaffected. This phenomenon is attributed to the innate immune system of ticks. The focus of this project is on stimulus-induced immunoreactive peptides known as antimicrobial peptides. In chapter 2, an attempt was made to identify a homolog of the anti Gram-positive and bacteriostatic peptide microplusin, in the salivary glands of the argasid tick Ornithodoros savignyi. It was reported previously that tissue and life stage specific expression of this transcript occurs in the fat body of adult, fully fed, female Rhipicephalus (Boophilus) microplus ticks. The positive control used for this study was unsuccessful due to the incorrect tissue and life stage of R. (B.) microplus ticks. No significant homolog was identified due to the possible existence of stringent regulation of expression as well as differences in the induction stimuli between argasid and ixodid ticks. Lysozyme catalyzes the cleavage of the β-1,4 glycosidic bond between N-acetyl muramic acid and N-acetyl glucosamine of the peptidoglycan layer of bacterial cell walls affording the molecule antibacterial activity. In argasid ticks, lysozyme was observed to be induced by feeding. In chapter 3, an attempt was made to elucidate the O. savignyi homolog of the O. moubata lysozyme molecule. The partial sequence obtained revealed the presence of a lysozyme homolog in O. savignyi. The tissue expression profile revealed constitutive expression in the midgut and ovaries and induction of transcription in the hemolymph upon feeding. In salivary glands, upregulation was observed following ingestion of Gram-positive bacteria. In chapter 4, the tissue expression profile of O. savignyi defensin was investigated. It was found that transcription is induced following the ingestion of Gram-positive bacteria, while in the hemolymph upregulation was observed upon feeding. Furthermore, chapter 4 saw the attempts made at the RNAi mediated silencing of the lysozyme and defensin transcripts. Silencing, analysed by real time PCR, was not efficient as no statistically significant silencing was observed. Observation of the phenotype revealed mortality. However, statistical analysis of silencing revealed that the mortality observed was not due to silencing, but non-specific and possibly the result of injury during injection. Overall, the abovementioned experiments revealed the tissue specificity of expression of ixodid microplusin and that a more strategic approach is required for the elucidation of the argasid homolog. The partial O. savignyi lysozyme sequence was elucidated together with the tissue expression profile of this molecule and O. savignyi defensin. The RNAi experiments require optimization for future studies. / Dissertation (MSc)--University of Pretoria, 2010. / Biochemistry / unrestricted

There is currently no independent monitoring and evaluation structure for state agricultural or veterinary services to support the “Batho Pele” principle of effective and efficient service delivery to the people of South Africa. Participatory rural appraisal was used in Moretele District, North West Province, to design, implement and assess veterinary extension on the tick and worm parasites of cattle. Veterinary extension, in contrast to agricultural extension, is defined as practical and understandable advice given to individual, groups, communities and population about livestock diseases and includes their prevention, treatment and control, as well as the way they influence the well being, health, and productivity of both humans and animals. The study was conducted in Moretele, which lies about 60km north of Pretoria, and is divided into three service delivery wards, each managed by an animal health technician. A random sample of 30 beef cattle farmers, each with a minimum of 10 cattle, was done in each ward. From each of these 90 farmers, five indicator cattle were purposively selected to include two calves, two sub-adults and one adult animal. Adult feeding ticks were sampled from predetermined sites and eggs per gram were estimated from pooled faecal samples of the same animals. The knowledge levels of animal health technicians (N=44) were assessed prior to the extension being given to the farmers and it was found to be inadequate. They were then given further training. Demographics and knowledge level of farmers were assessed using structured interviews. Baseline sampling for parasites was done on the indicator cattle. A farmer’s day and monthly extension using the visit and training method of extension was done with the farmers over a period of 12 months. The level of knowledge of the farmers was reassessed and the indicator cattle resampled at the same time of the year as the first sampling. It was found that although there was a significant increase in the farmers knowledge, there were no significant differences in the level or species of parasites. It was concluded that animal health technicians did not normally have sufficient knowledge of the subject to give farmers affective extension messages. It was also concluded that knowledge and implementation of extension are not the same thing and that further research is required into the reasons for lack of implementation. / Dissertation (MSc (Veterinary Sciences))--University of Pretoria, 2004. / Paraclinical Sciences / unrestricted

6 1. Abstrakt Hyaluronidázy jsou významnou skupinou enzymů odpovědných za štěpení kyseliny hyaluronové, která je jednou z hlavních složek pojivové tkáně obratlovců. U většiny krevsajících členovců je tento enzym přítomen ve slinách a usnadňuje sání tím, že se podílí na průniku kůží a zvětšení potravní léze v místě bodnutí. Vzniklé fragmenty extracelulární matrix mohou navíc modulovat lokální imunitní odpověď hostitele a zvyšovat pravděpodobnost přenosu patogenů. V naší práci jsme se zaměřili na průkaz hyaluronidázové aktivity u klíštěte Ixodes ricinus (Acari: Ixodidae) a zástupců čtyř rodů ovádů (Diptera: Tabanidae). V slinných žlázách klíšťat se hyaluronidázovou aktivitu detekovat nepodařilo. Naopak, ve slinných žlázách ovádů jsme prokázali velmi silnou aktivitu u všech studovaných druhů a dále ji charakterizovali biochemickými a elektroforetickými metodami. Hyaluronidázy ovádů štěpí hyaluronan i chondroitin sulfát; enzymy jednotlivých druhů se mírně liší molekulovou hmotností, pH optimem a citlivostí k redukujícím podmínkám. Abstract (in English) Hyaluronidases are an important group of enzymes responsible for cleaving hyaluronic acid, which is a major component of the extracellular matrix of vertebrates. In bloodsucking arthropods these enzymes are frequently present in saliva. Salivary hyaluronidases...

Considerable research on ticks has been done since it was discovered that the Rocky Mountain wood tick, Dermacentor andersoni Stiles, was a principal vector of Rocky Mountain spotted fevero Additional disease agents of man are also transmitted by D. andersoni. This tick and a closely related species, Dermacentor parumapertus Neuman, occur commonly in Utah. The two species are often closely associated, although D. andersoni is believed to occur in the mountains, whereas D. parumapertus is in the desert valleys. Diseases affecting animals in nature are transmitted by ticks of both species. Consequently, any interaction between the two may be influential in the maintenance of diseases in nature communicable to man and his domestic animals.