Made available in DSpace on 2016-04-28T20:15:24Z (GMT). No. of bitstreams: 1 2007-Raquel Moreira Pires dos Santos Melo.pdf: 931840 bytes, checksum: 8bf91d313bc9957a8e14c9fcb0238d3c (MD5) Previous issue date: 2007-02-27 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / The purpose of this study is to follow up the morphology and biology of Rhipicephalus sanguineus after being submitted to the arthropods growth regulator fluazuron on rabbits. This study took place in the dependence of the Laboratories of Development of Parasiticides Products (LDPP) and Acari Morfophisiology (LAM), both part of the Department of Animal Parasitology of Veterinary Institute of Universidade Federal Rural do Rio de Janeiro (UFRRJ), in Serop?dica, (22?44'38"S, 43?42'27"W). The growth regulators represents a new category that has been employed as ectoparasiticide on pets. This chemical group do not presents a knockdown effect, but does act slowly and gradually interfering on parasite s growth and development. The AGRs are classified as per their mechanism of action. The first group is a juvenile hormone analogue, benzoilphenil-urea inhibiting the chitin synthesis. The second are the inhibitors of deposition of chitin, triazin s and pyrimidin s derived. The FZN was the first growth regulator registered for ixodides tick s control. Four phases were analyzed; each one using ten rabbits divided in control and treated. At phase I (larvae stage), the animals were infested with R. sanguineus larvae on the ratio of 2500 larvae/rabbit. At phase II (nymph stage), with the ratio of 200 nymphs/rabbit. At phase III (adults), the animals were infested with 25 males and 25 females/rabbit. At phase IV, the infestation was done with 2500 larvae from engorged female ticks recovered on phase III. Fluazuron was applied as a pour on formulation on each phase using 10mg/kg. The experimentations during the parasitic phase had been carried through in ambient conditions, the period of August the November of 2006, and of the not parasitic phase in controlled conditions of laboratory (27 ? 1 ?C and 80 ? 10% UR, in escotofase). Parasitic and not parasitic stages of larvae, nymphs and adults were evaluated in different parameters. The FZN changed the biology of R. sanguineus, such as: increasing the pre-posture period; reducing the weight of eggs mass; increasing the incubation period of eggs; reducing the rate of reproductive efficiency, inhibiting the ecdyse process from larvae to nymph and from nymph to adult, efficacy in the inhibition of female reproduction and all recovered specimen from all the stages in the treated group showed sensible tegument, elliptical body and lethargic behavior. It is concluded that fluazuron employed in the dose of 10mg/kg of corporal weight in rabbits promoted alterations morphologies, biological and mannering in R. sanguineus that they indicate the possibility of job of this AGR in the control of this carrapato, with the particularitity in the reduction of the reinfesta??o tax. However new studies need to be carried through aiming at the effectiveness of fluazuron in the control of R. sanguineus when used in dogs. The use of rabbits was a preliminary test to indicate or not it activity of fluazuron on sanguineus carrapato R. sanguineus. / A realiza??o deste estudo teve como objetivo acompanhar a morfologia e biologia de Rhipicephalus sanguineus submetidos ao regulador de crescimento de artr?podes (AGR s), fluazuron, (FZN) em coelhos. O trabalho foi realizado nas depend?ncias dos Laborat?rios de Desenvolvimento de Produtos Parasiticidas (LDPP) e de Morfofisiologia de ?caros (LMA), ambos pertencentes ao Departamento de Parasitologia Animal do Instituto de Veterin?ria da Universidade Federal Rural do Rio de Janeiro (UFRRJ), localizada no munic?pio de Serop?dica (latitude 22?44'38" sul, longitude 43?42'27" oeste). Os reguladores de crescimento representam uma nova categoria que vem sendo empregada amplamente no controle de ectoparasitos de pequenos animais. Este grupo qu?mico n?o apresenta efeito knockdown , atua de forma lenta e gradual interferindo no seu crescimento e desenvolvimento. Os AGR s foram divididos de acordo com o seu mecanismo de a??o, em an?logo s do horm?nio juvenil, benzoilfenil ur?ias - atuando na inibi??o da s?ntese de quitina e inibidores da deposi??o de quitina, derivados da triazina e da pirimidina. O FZN foi o primeiro regulador de crescimento a ser registrado para o controle de carrapatos ixod?deos O experimento foi dividido em 4 etapas, sendo que em cada uma delas utilizados 10 coelhos, divididos em grupos controle e tratado. Na etapa I (fase de larva) os coelhos foram infestados com larvas de R. sanguineus na raz?o de 2500 larvas/coelho. Na etapa II (fase de ninfa), na raz?o de 200 ninfas/coelho. Na etapa III (adultos), os coelhos foram infestados com 25 machos e 25 f?meas/coelho. Na etapa IV, os coelhos foram infestados com 2500 larvas oriundas das tele?ginas recuperadas na etapa III. A dose de FZN aplicada sob a forma pour-on em todas as etapas foi de 10mg/kg. As experimenta??es durante a fase parasit?ria foram realizadas em condi??es ambientais, no per?odo de agosto a novembro de 2006, e da fase n?o parasit?ria em condi??es controladas de laborat?rio (27 ? 1 ?C e 80 ? 10% UR, em escotofase). Foram analisados par?metros de fase parasit?ria e n?o parasit?ria, para larvas, ninfas e adultos. O FZN causou altera??es na biologia de R. sanguineus tais como: aumento no per?odo de pr?-postura, diminui??o no peso da massa de ovos, aumento no per?odo de incuba??o dos ovos, diminui??o do ?ndice de efici?ncia reprodutiva, inibi??o do processo de ecdise entre os est?gios de larva para ninfa e de ninfa para adulto, efic?cia na inibi??o da reprodu??o das f?meas e todos os est?gios recuperados do grupo tratado tinham tegumento sens?vel, corpo el?ptico e comportamento let?rgico. Conclui-se que o fluazuron empregado na dose de 10mg/kg de peso corporal em coelhos promoveu altera??es morfologias, biol?gicas e comportamentais em R. sanguineus que indicam a possibilidade de emprego deste AGR no controle desse carrapato, com a particularidade na redu??o da taxa de reinfesta??o. Entretanto novos estudos necessitam ser realizados visando a efic?cia do fluazuron no controle de R. sanguineus quando empregado em c?es. O uso de coelhos foi um teste preliminar para indicar ou n?o a atividade do fluazuron sobre o carrapato R. sanguineus.

Made available in DSpace on 2016-04-28T20:15:26Z (GMT). No. of bitstreams: 1 2007-Marcos Pinheiro Franque.pdf: 2601170 bytes, checksum: cef61cb976b6652b0e5f4fe212f81de9 (MD5) Previous issue date: 2007-02-28 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico / This work aimed the evaluation of biological parameters of parasitic and non-parasitic phases of Rhipicephalus (Boophilus) microplus (= Boophilus microplus) in equine. Four equines were individually infested with approximately 40,000 larvae obteined of R. (B.) microplus females engorged in bovines, being in two of them established three infestations. In the place of larvae fixation, it was noticed development of an intense itchy reaction and the larvae involved in a serum exudates. A larval mortality of approximately 90% was observed, with a small number of larvae changing to nymph stage. The mortality of nymphs was around 60%, with reduction of the itchy reaction, and in the adult stage was observed approximately 30% of mortality. As for the susceptibility, it was observed that two equine were resistant, one moderately resistant and one sensitive to the establishment of infestation by R. (B.) microplus. In the equine considered sensitive, were made observations of parasitic and non-parasitic phase of this ixodid. According to the parameters of the parasitic phase of R. (B.) microplus, of the three experimental infestations, the day at the beginning of detachment of females occurred among infestations were 28 and 31 days, during 12 days at first infestation and 20 days at second and thirst infestation, presentin modal day at 32?; 36? and 37? day, respectively. Were recovered 179 females of R. (B.) microlus at first infestation, 187 and 358 at the second and thirst infestation, corresponding to a mean recovery rate between 0.90 and 1.79%. The means periods of parasitic phase increase among infestations, during between 33. 27 and 38.51 days, being obteined females with mean weight of 90.0mg at the first infestation, 81.5 mg at second and 109.4mg at thirst infestation, considering all females recovered. For the estudy of the non-parasitic phase were selected form each infestation 20, 49, 71 females, presenting means weights of 151.8; 121.1 and 147.8 mg, respectively. The means periods of pre-posture were between 2.8 and 3.5 days. The means periods of posture were of 13.6; 11.7 and 13.4 days, respectively, with pick of posture occurring at the 3rd day after the beginning at three infestations. The means weights of posture were verified between 57.3 and 80.6mg corresponding to 1,146.9 and 1,611.4 eggs produced. The means periods of incubation of eggs produced by R. (B.) microplus studied females occurred between 23.7 and 29 days, presenting hatch means rates decreasing of 67% in the first infestation to 54.7% at the thirst infestation. The means of reprodutive efficiency index also decreased among infestations of 35.5% to 26.9% at the last infestation. It was verified that the means periods between the infestation date and the beginning of the larval appearance were between 60.0 and 64.9 days. These results demonstrate that, starting from experimental infestation in equine, R. (B.) microplus is able to complete the biological cycle for at least one generation, resulting in a number of larvae enough to infest pastures. / Este trabalho teve como objetivo avaliar par?metros biol?gicos de Rhipicephalus (Boophilus) microplus (= Boophilus microplus) mediante infesta??es expermentais em eq?inos. Foram utilizados quatro eq?inos infestados individualmente com aproximadamente 40.000 larvas, obtidas de f?meas de R. (B.) microplus alimentadas em bovino, sendo realizadas tr?s infesta??es em dois destes equinos. No local de fixa??o das larvas notou-se desenvolvimento de uma intensa rea??o pruriginosa e as larvas envolvidas em um exsudato seroso. Foi observada uma mortalidade larval de aproximadamente 90%, com um pequeno n?mero larvas mudando para o est?gio de ninfa. A mortalidade de ninfas ocorreu em torno de 60%, com redu??o da rea??o pruriginosa, e no est?gio adulto notou-se mortalidade de aproximadamente 30%. Quanto ? susceptibilidade, observou-se que dois eq?inos foram resistentes, um moderadamente resistente e um sens?vel ao estabelecimento da infesta??o por R. (B.) microplus. No eq?ino considerado sens?vel, foram realizadas as observa??es de fase parasit?ria e n?o parasit?ria deste ixod?deo. Em rela??o aos par?metros da fase parasit?ria de R. (B.) microplus, nas tr?s infeta??es, o dia de in?cio do desprendimento das f?mas ocorreu entre 28 e 31 dias, durando 12 dias na primeira infesta??o e 20 dias na segunda e terceira infesta??es, apresentando dia modal no 32?, 36? e 37? dia respectivamente, ap?s as infesta??es com as larvas. Foram recuperadas 179 f?meas de R. (B.) microplus na primeira infesta??o, 187 na segunda e 358 na terceira infesta??o, correspondendo a uma taxa de recupera??o entre 0,90 e 1,79%. O per?odo m?dio da fase parasit?ria aumentou entre a primeira e terceira infesta??es, ocorrendo entre 33,27 e 38,51 dias, verificando-se f?meas com peso m?dio de 90,0mg na primeira infesta??o, 81,5mg na segunda e 109,4mg na terceira infesta??o, considerando-se todas as f?meas recuperadas. Para o estudo da fase n?o parasit?ria foram selecionadas respectivamente 20, 49 e 71 f?meas de cada infesta??o que apresentaram peso m?dio, respectivamente, de 151,8mg; 121,1 mg e 147,8mg. Observou-se um per?odo m?dio de pr?-postura entre 2,8 e 3,5 dias. O per?odo m?dio de postura foi de 13,6; 11,7 e 13,4 dias, respectivamente, entre as infesta??es, com pico de postura ocorrendo no 3? dia ap?s seu in?cio. Verificou-se um peso m?dio de postura entre 57,3 a 80,6mg, nas infesta??es, o que corresponde ? produ??o m?dia de ovos entre 1.146,9 e 1.611,0. O per?odo m?dio de incuba??o dos ovos das f?meas estudadas de R. (B.) microplus, durou entre 23,7 e 29 dias, apresentando uma taxa de eclos?o m?dia decrescendo de 67% na primeira infesta??o, para 54,7% na terceira infesta??o. O ?ndice m?dio de efici?ncia reprodutiva tamb?m decresceu ente as infesta??es de 35,5%, para 26,9% na ?ltima infesta??o. Verificou-se que o per?odo m?dio entre a data de infesta??o e o in?cio da eclos?o larval ocorreu entre 60 e 64,9 dias. Estes resultados demonstram que, a partir de infesta??es experimentais em eq?inos, R. (B.) microplus ? capaz de completar seu ciclo biol?gico por pelo menos uma gera??o, resultando em um n?mero de larvas suficiente para infestar parstagens.

Made available in DSpace on 2016-04-28T20:15:28Z (GMT). No. of bitstreams: 1 2000- Flavia Santos Pinto.pdf: 229877 bytes, checksum: 5ac9dc34947f0f90f514886c5e4e15e0 (MD5) Previous issue date: 2000-02-28 / The natural sensitivity of two breeds of rabbit, White New Zealand (WNZ) e California (CA), and their crossbreeds (CB) to Rhipicephalus sanguineus were compared under experimental conditions. Three rabbits of each breed, without previously tick exposure, were infested once with 2300 larvae, 100 nymphs and 40 adults (20 males + 20 females). The experiment with the parasitic phase was conducted in the environment while the free-living phase was conducted under 27 ? 1? C, 80 ? 10 % relative humidity and scotophase. The dynamic of the sensitivity had in the larvae phase a variation statistically significant (p<0.05) in the pr?ecdise period, with the best performance in the CB group. However, the percentage of recovery was greater in the treatment of WNZ. In the adult phase, females of the WNZ group presented a greater mean weight (p<0.0) e did not differ from the Nutritional Efficiency Index(NEI) from CB, which had the best performance. For larvae of the females in the three treatments the only statistic variation (p<0.05) was with the period hatching, where the CA group presented the smallest period, the CB group presented the greatest and the WNZ group did not change statistically in the both. The life cycle in all three groups was very close, without great differences according to the treatment received. / A sensibilidade natural a Rhipicephalus sanguineus de duas ra?as de coelhos, Nova Zel?ndia e Calif?rnia, e seus mesti?os foi comparada em condi??es experimentais. Tr?s coelhos de cada ra?a, sem contato pr?vio com carrapatos, foram infestados uma ?nica vez com 2300 larvas, 100 ninfas e 40 adultos (20 machos + 20 f?meas). O experimento com a fase parasit?ria foi conduzido no meio ambiente enquanto que o da fase de vida livre foi conduzido a 27 ? 1? C, 80 ? 10 % de umidade relativa e escotofase. A din?mica da sensibilidade obteve na fase larval uma varia??o estatisticamente significativa ( p< 0,05) no per?odo de pr?-ecdise com melhor desempenho no grupo Mesti?o, por?m o percentual de recupera??o foi maior no tratamento Nova Zel?ndia. N?o houve diferen?a estatisticamente significativa (p> 0,05) na fase de ninfas, onde o grupo de melhor desempenho foi o Calif?rnia. Na fase adulta , as f?meas do grupo Nova Zel?ndia apresentaram maior peso m?dio(p<0,05) e n?o diferiram para o ?ndice de Efici?ncia Nutricional do tratamento Mesti?o, o qual teve melhor desempenho. Para larvas oriundas das f?meas que passaram pelos tr?s tratamentos a ?nica varia??o estat?stica (p<0,05) foi com o per?odo de eclos?o, onde o grupo Calif?rnia apresentou menor per?odo, o grupo Mesti?o o maior e Nova Zel?ndia n?o variou estatisticamente destes dois. O ciclo biol?gico nos tr?s grupos foi muito pr?ximo, sem grandes diferen?as de acordo com o tratamento recebido.

Made available in DSpace on 2016-04-28T20:15:28Z (GMT). No. of bitstreams: 1 2008- Leonardo Burlini Soares.pdf: 1364725 bytes, checksum: a5a09ed4e869fe7e3a75342f6f3f7d8a (MD5) Previous issue date: 2008-02-28 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / The taxonomic status of ticks of the Rhipicephalus sanguineus group is difficult to be determined by phenotypic methods and has been object of frequent revisions and ongoing debate. The differentiation of species within this group has clinical importance, especially in veterinary medicine due to transmission of different pathogens. The objective of this study was to evaluate de genetic variability of Rhipicephalus sanguineus from different geografic regions of Brazil and fill in some gaps in the species study. This work was conducted in the laboratories of: Ixodology of the W. O. Neitz station for parasitologic research, Molecular Biology and Acarology, of the Departamento de Parasitologia Animal, Instituto de Veterin?ria, da Universidade Federal Rural do Rio de Janeiro; and in the laboratories of: Genetic and Biochemistry, and Genome of EMBRAPA Agrobiologia (RJ). Mitochondrial DNA (mt-DNA) fragments of R sanguineus from Esp?rito Santo (ES), Goi?s (GO), Par? (PA), Rond?nia (RO), Mato Grosso do Sul (MS), Rio de Janeiro (RJ) e Rio Grande do Norte (RN) were compared. The results showed that fasting larvae, preserved in ethanol, from the different localities presented genotipic variability considering the 12S and 16S mt-rDNA genes. To 12S, the Esp?rito Santo strain presented the highest genetic diversity in comparison with the other brazilian strains; to 16S, the distinction can be given to the Rio de Janeiro strain, which presented the highest divergence values in regarding the other strains. The intraspecific variability detected between the isolates ranged from 0 to 6.6% regarding the 12S gene and from 0 to 2.7% regarding the 16S. On the other hand, a strong genetic relationship was detected between brazilian isolates and asian R. sanguineus (0-5.8% of variability for 12S, and 0-1.3% for 16S) and between the brazilian strains and african Rhipicephalus turanicus (2.2-7.6% of difference to 12S) while R. sanguineus populations from Argentina and Uruguai appeared to be related to French R. sanguineus (identic 12S sequences); considering 16S, R. sanguineus from Israel presented moderate distance from the brazilian isolates (3.6-5.8%). These results showed that the differences between these brazilian isolates are great and to a certain extent greater than should be expected between some of them, and that the systematic of R. sanguineus ticks from Latin America as much as of countries of other continents should be better and more studied. Wide variations, such as these might account for the reported worldwide differences in several biological parameters in this species. / O posicionamento taxon?mico de carrapatos do grupo Rhipicephalus sanguineus ? de dif?cil determina??o por m?todos fenot?picos e tem sido objeto de freq?entes revis?es e debates cont?nuos. A diferencia??o das esp?cies dentro desse grupo ? de import?ncia cl?nica, especialmente em medicina veterin?ria, devido a transmiss?o de diferentes pat?genos. O estudo teve como objetivo avaliar a variabilidade gen?tica de Rhipicephalus sanguineus provenientes de diferentes regi?es geogr?ficas do Brasil e preencher algumas lacunas no estudo da esp?cie. Este trabalho foi realizado nos laborat?rios de: Ixodologia da Esta??o para Pesquisas Parasitol?gicas W. O. Neitz, Biologia Molecular e Acarologia, pertencentes ao Departamento de Parasitologia Animal, Instituto de Veterin?ria, da Universidade Federal Rural do Rio de Janeiro; e nos laborat?rios de: Gen?tica e Bioqu?mica, e Genoma da EMBRAPA Agrobiologia (RJ). Foram comparados fragmentos de DNA mitocondrial (DNA-mt) de R sanguineus do Esp?rito Santo (ES), Goi?s (GO), Par? (PA), Rond?nia (RO), Mato Grosso do Sul (MS), Rio de Janeiro (RJ) e Rio Grande do Norte (RN). Os resultados mostram que larvas em jejum, preservadas em etanol, das diferentes localidades apresentaram variabilidade genot?pica considerando-se os genes 12S e 16S DNAr-mt. Para o gene 12S DNAr-mt, a amostra do Esp?rito Santo apresentou a maior diversidade gen?tica em compara??o ?s demais amostras brasileiras; j? para o gene 16S DNAr-mt, o destaque pode ser dado ? amostra do Rio de Janeiro, que apresentou os maiores valores de diverg?ncia em rela??o ?s demais seq??ncias. A variabilidade intraespec?fica detectada entre os isolados variou de 0 a 6,6% em rela??o ao gene 12S DNAr-mt e de 0 a 2,7% em rela??o ao 16S DNArmt. Por outro lado, uma forte rela??o gen?tica foi detectada entre os isolados brasileiros e R. sanguineus asi?ticos (0-5,8% de variabilidade para 12S, e 0-1,3% para 16S DNAr-mt) e entre as amostras brasileiras e Rhipicephalus turanicus africanos (2,2-7,6% de diferen?a para 12S) enquanto popula??es de R. sanguineus da Argentina e Uruguai se relacionaram com R. sanguineus da Fran?a (seq??ncias de 12S id?nticas); no tocante ao 16S, R. sanguineus de Israel apresentou moderada dist?ncia dos isolados brasileiros (3,6-5,8%). Esses resultados mostraram que as diferen?as entre esses isolados brasileiros s?o grandes e at? mesmo maiores do que poderia se esperar entre alguns deles, e que a sistem?tica de carrapatos R. sanguineus, tanto da Am?rica Latina quanto de pa?ses de outros continentes deve ser melhor e mais estudada. Amplas varia??es como estas podem justificar as diferen?as mundiais relatadas para diversos par?metros biol?gicos nessa esp?cie.

Submitted by Celso Magalhaes (celsomagalhaes@ufrrj.br) on 2017-05-03T17:33:38Z No. of bitstreams: 1 2012 - Hermes Ribeiro Luz.pdf: 2629217 bytes, checksum: 8e75ef38df8d873d63a0480f410610a8 (MD5) / Made available in DSpace on 2017-05-03T17:33:38Z (GMT). No. of bitstreams: 1 2012 - Hermes Ribeiro Luz.pdf: 2629217 bytes, checksum: 8e75ef38df8d873d63a0480f410610a8 (MD5) Previous issue date: 2012-02-09 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico - CNPq / This study analyzes the prevalence and mean intensity of infestation of ticks on birds captured in ESEC-Pirapitinga (180 20?S e 450 17?WGr), in Tr?s Marias, Minas Gerais, Brazil. Of a total of 1.295 birds captured, distributed in nine orders, 28 families and 92 species, 165 (12.7%) individuals were parasitized by ticks. Of these 160 (97%) belonged to the order Passeriformes and only 5 (3%) belonged to the grouping of non-passerines. We identified five tick species: Amblyomma longirostre (n = 274) was the most common species followed by Amblyomma parvum (n = 43), Amblyomma nodosum (n = 39), Amblyomma ovale (n = 24) and Rhipicephalus sanguineus (n = 7). The average infestation was 2.7 ticks by bird. Only 16 (10%) birds were infected with more than 5 ticks, while 97 (58.8%), birds were infected with a single tick. Also present in this study, some new records of A. longirostre. A. parvum, A. nodosum, A. ovale and R. sanguineus parasitizing birdscn / No presente estudo analisamos a preval?ncia e intensidade m?dia de infesta??o de carrapatos em aves capturadas na ESEC-Pirapitinga (180 20?S e 450 17?WGr), no munic?pio de Tr?s Marias, Minas Gerais, Brasil. De um total de 1295 aves capturadas, distribu?das em 9 ordens, 28 fam?lias e 92 esp?cies, 165 (12,7%) indiv?duos estavam parasitados por carrapatos. Destes 160 (97%) pertencia a ordem Passeriformes e apenas 5 (3%) pertenciam ao grupamento dos n?o passeriformes. Foram identificadas 5 esp?cies de carrapatos: Amblyomma longirostre (n= 273) foi a esp?cie mais comum seguida por Amblyomma parvum (n=43), Amablyomma nodosum (n=39), Amblyomma ovale (n=24) e Rhipicephalus sanguineus (n=7). A infesta??o m?dia foi de 2,7 carrapatos por ave. Somente 16 (10%) aves estavam infestadas por mais que 5 carrapatos, enquanto que 97(58,8%), aves estavam infestadas por um ?nico carrapato. Tamb?m apresentamos, neste estudo, alguns registros novos de A. longirostre. A. parvum, A. nodosum, A. ovale e R. sanguineus parasitando diferentes esp?cies de aves

Orientador: Maria Izabel Camargo Mathias / Coorientador: Patrícia Rosa de Oliveira / Banca: Gervasio Henrique Bechara / Banca: Erik Daemon de Souza Pinto / Resumo: Carrapatos são ectoparasitas obrigatoriamente hematófagos que podem transmitir diversas doenças aos hospedeiros durante o seu processo de alimentação. Ao lesionar mecanicamente o tecido acabam induzindo respostas inflamatórias na pele, local onde se fixam. Na tentativa de minimizar a ação destes sobre os hospedeiros tem-se recorrido ao controle químico por meio da utilização de diversos produtos, dentre estes a selamectina, princípio ativo de muitos acaricidas, inclusive do Revolution® (Pfizer), uma lactona macrocíclica capaz de causar danos neurotóxicos no carrapato. Foi objetivo deste trabalho analisar, por meio de técnicas histológica, histoquímica, detecção da atividade da fosfatase ácida (enzimas hidrolíticas responsáveis pela detecção de morte celular autofágica) e microscopia confocal de varredura a laser, a ocorrência de alterações morfofisiológicas na pele de coelhos hospedeiros expostos à selamectina e infestados por Rhipicephalus sanguineus adultos. Os coelhos (n=12) foram expostos ao produto nas concentrações de 100% (grupo de tratamento I - GTI) e de 80% (grupo de tratamento II - GTII) e posteriormente infestados apresentaram no exame histológico diminuição da espessura da camada córnea do epitélio, o qual sofreu diminuição da quantidade de camadas celulares (adelgaçamento), processo este acompanhado pela formação acentuada de edemas subepidérmicos (com aumento de exudato), o que provocou a desorganização das fibras colágenas do tecido conjuntivo da camada dérmica. Os testes histoquímicos revelaram forte marcação PAS positiva nos folículos pilosos e em algumas regiões da derme e ressíntese de fibras colágenas demonstrada por meio da técnica do tricômico de Mallory. Os resultados obtidos para a detecção da atividade da fosfatase ácida nos indivíduos expostos às... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Ticks are ectoparasites obligatorily hematophagous that can transmit various diseases to their hosts during the feeding process. By mechanically injured in the tissue eventually inducing inflammatory responses in the skin where they attach. In an attempt to minimize their action on the host has recourse to chemical control through the use of various acaricides, among these selamectin, the active principle of many acaricides, including the Revolution ® (Pfizer), a macrocyclic lactone capable of causing neurotoxic damage. This study aimed to analyze, using histological techniques, histochemical, detection of acid phosphatase activity (hydrolytic enzymes responsible for the detection of autophagic cell death) and confocal laser scanning, the occurrence of morphophysiological changes in the skin of hosts rabbits exposed to the selamectin and infested by adults of Rhipicephalus sanguineus. The rabbits (n=12) exposed to the product at concentration of 100% (treatment group I - TGI) and 80% (treatment group II - TGII) and infested showed, in histologic examination, a partial and/or complete decrease in the stratum corneum of epithelium, which suffered a decrease in the amount of cell layers with consequent reduction of stratification (thinning) of the epithelium and marked subepidermal edema formation (with increase of exudates) which caused the disorganization of the collagen fibers of the connective tissue of the dermal layer. Histochemical tests showed strong staining PAS positive in hair follicles and in some regions of the dermis, and the resynthesis of collagen fibers has been demonstrated by Mallory trichrome technique. The confocal images corroborate the results of the histological analysis, showing the thinning of the epithelium of the individuals' skin of the treatment groups I and II (TGI and TGII), where this layer... (Complete abstract click electronic access below) / Mestre

THESIS ABSTRACT Current control strategies for livestock ectoparasites are limited by problems associated with chemical resistance and residues. Fungal biopesticides could provide an alternative control without these problems. However, a strategic approach is needed to first evaluate the suitability of selected fungal isolates for fungal biopesticide development. Two ectoparasites of significance to cattle and sheep are the cattle tick Rhipicephalus (Boophilus) microplus (Canestrini) and the Australian sheep blowfly Lucilia cuprina (Wiedmann). The fungus Metarhizium anisopliae (Metsch.) Sorokin) was evaluated for its potential to control these livestock ectoparasites. The growth characteristics of 30 isolates of M. anisopliae were investigated. Radial growth measurements were used to identify vigorous isolates that grew well at 30C and were capable of growing at 35C. A qualitative assessment of sporulation capacity further refined the candidate isolate group. A possible nutritive role of oil in the formulation was also investigated. However, there was no clear support for the theory that oil as a formulation additive could boost the germination and growth of the fungal conidia in vitro. Quantal response bioassays were conducted with cattle ticks and sheep blowflies using a range of conidial doses of three different isolates of M. anisopliae and different methods of inoculation. Ticks were either dosed with 2 µl or immersed in the conidial doses. Blowflies were either dosed with 2 µl of the conidial doses or fed conidia mixed with sugar. Probit analyses were carried out on the mortality data to compare the virulence of these isolates to ticks and blowflies and look for indications of different virulence mechanisms employed by M. anisopliae isolates when invading these hosts. One isolate (ARIM16) showed high virulence to both hosts killing 95 % of ticks after two days and 88 (±2) % of blowflies after four days. Strikingly different mortality patterns indicated quite different virulence mechanisms operating when M. anisopliae invades ticks or blowflies. The mortality pattern seen with ticks suggested that the number of conidia adhering per unit area of the cuticle was more important for rapid tick death than the total number of conidia contacting the entire tick surface. Blowflies fed conidia mixed with food died rapidly after an initial lag phase regardless of dose. Microscopic investigations were carried out to resolve the basis of the virulence patterns observed. The spatial and temporal aspects of the invasion of ticks and blowflies by M. anisopliae isolate ARIM16 were investigated with different types of microscopy. The scanning electron microscope and stereo light microscope were used to record surface changes and events and the compound light microscope revealed internal changes. Two distinctly different patterns of invasion were found in ticks and blowflies. Fungal conidia germinated on the surface of ticks then hyphae simultaneously penetrated into the tick body and grew across the tick surface. There was extensive fungal degradation of the tick cuticle with a preference for the outer endocuticle. While large numbers of conidia adhered to the surface of blowflies, no conidia were recorded germinating on external surfaces. One germinating conidium was seen in the entrance to the buccal cavity. Investigations of the fly interior revealed a higher density of hyphal bodies in the haemolymph surrounding the buccal cavity than in haemolymph from regions of the upper thorax. This pattern suggested that fungal invasion of the blowfly is through the buccal cavity. Plentiful extracellular mucilage was seen around the hyphae on ticks, and crystals of calcium oxalate were seen amongst the hyphae on the surface of ticks and in the haemolymph of blowflies killed by M. anisopliae isolate ARIM16. It was considered that cattle ticks are more suited for control with fungal biopesticides than adult blowflies. Three field trials were conducted over twelve months to assess the pathogenicity of M. anisopliae to parasitic stages of R. microplus on dairy heifers under different environmental conditions. Two isolates were selected based on their high optimal growth temperature (30oC), good conidial production characteristics and ability to kill adult engorged ticks in the laboratory in minimum time. Conidia were formulated in an oil emulsion and applied using a motor driven spray unit. Surface temperatures of selected animals were monitored, as were the ambient temperature and relative humidity. Unengorged ticks sampled from each animal immediately after treatment were incubated under laboratory conditions to assess the efficacy of the formulation and application. Egg production by engorged ticks collected in the first 3 days after treatment was monitored. Side counts of standard adult female ticks were conducted daily, before and after treatment to assess the performance of the fungus against all tick stages on the animals. At each trial the formulation caused 100% mortality in unengorged ticks that were removed from cattle and cultured under laboratory conditions. A significant reduction in egg production was recorded for engorged ticks collected in the three days post treatment. In the field, the fungal formulation had an inconsistent effect on ticks, which might be due to the influence of environmental temperature and humidity.

THESIS ABSTRACT Current control strategies for livestock ectoparasites are limited by problems associated with chemical resistance and residues. Fungal biopesticides could provide an alternative control without these problems. However, a strategic approach is needed to first evaluate the suitability of selected fungal isolates for fungal biopesticide development. Two ectoparasites of significance to cattle and sheep are the cattle tick Rhipicephalus (Boophilus) microplus (Canestrini) and the Australian sheep blowfly Lucilia cuprina (Wiedmann). The fungus Metarhizium anisopliae (Metsch.) Sorokin) was evaluated for its potential to control these livestock ectoparasites. The growth characteristics of 30 isolates of M. anisopliae were investigated. Radial growth measurements were used to identify vigorous isolates that grew well at 30C and were capable of growing at 35C. A qualitative assessment of sporulation capacity further refined the candidate isolate group. A possible nutritive role of oil in the formulation was also investigated. However, there was no clear support for the theory that oil as a formulation additive could boost the germination and growth of the fungal conidia in vitro. Quantal response bioassays were conducted with cattle ticks and sheep blowflies using a range of conidial doses of three different isolates of M. anisopliae and different methods of inoculation. Ticks were either dosed with 2 µl or immersed in the conidial doses. Blowflies were either dosed with 2 µl of the conidial doses or fed conidia mixed with sugar. Probit analyses were carried out on the mortality data to compare the virulence of these isolates to ticks and blowflies and look for indications of different virulence mechanisms employed by M. anisopliae isolates when invading these hosts. One isolate (ARIM16) showed high virulence to both hosts killing 95 % of ticks after two days and 88 (±2) % of blowflies after four days. Strikingly different mortality patterns indicated quite different virulence mechanisms operating when M. anisopliae invades ticks or blowflies. The mortality pattern seen with ticks suggested that the number of conidia adhering per unit area of the cuticle was more important for rapid tick death than the total number of conidia contacting the entire tick surface. Blowflies fed conidia mixed with food died rapidly after an initial lag phase regardless of dose. Microscopic investigations were carried out to resolve the basis of the virulence patterns observed. The spatial and temporal aspects of the invasion of ticks and blowflies by M. anisopliae isolate ARIM16 were investigated with different types of microscopy. The scanning electron microscope and stereo light microscope were used to record surface changes and events and the compound light microscope revealed internal changes. Two distinctly different patterns of invasion were found in ticks and blowflies. Fungal conidia germinated on the surface of ticks then hyphae simultaneously penetrated into the tick body and grew across the tick surface. There was extensive fungal degradation of the tick cuticle with a preference for the outer endocuticle. While large numbers of conidia adhered to the surface of blowflies, no conidia were recorded germinating on external surfaces. One germinating conidium was seen in the entrance to the buccal cavity. Investigations of the fly interior revealed a higher density of hyphal bodies in the haemolymph surrounding the buccal cavity than in haemolymph from regions of the upper thorax. This pattern suggested that fungal invasion of the blowfly is through the buccal cavity. Plentiful extracellular mucilage was seen around the hyphae on ticks, and crystals of calcium oxalate were seen amongst the hyphae on the surface of ticks and in the haemolymph of blowflies killed by M. anisopliae isolate ARIM16. It was considered that cattle ticks are more suited for control with fungal biopesticides than adult blowflies. Three field trials were conducted over twelve months to assess the pathogenicity of M. anisopliae to parasitic stages of R. microplus on dairy heifers under different environmental conditions. Two isolates were selected based on their high optimal growth temperature (30oC), good conidial production characteristics and ability to kill adult engorged ticks in the laboratory in minimum time. Conidia were formulated in an oil emulsion and applied using a motor driven spray unit. Surface temperatures of selected animals were monitored, as were the ambient temperature and relative humidity. Unengorged ticks sampled from each animal immediately after treatment were incubated under laboratory conditions to assess the efficacy of the formulation and application. Egg production by engorged ticks collected in the first 3 days after treatment was monitored. Side counts of standard adult female ticks were conducted daily, before and after treatment to assess the performance of the fungus against all tick stages on the animals. At each trial the formulation caused 100% mortality in unengorged ticks that were removed from cattle and cultured under laboratory conditions. A significant reduction in egg production was recorded for engorged ticks collected in the three days post treatment. In the field, the fungal formulation had an inconsistent effect on ticks, which might be due to the influence of environmental temperature and humidity.

The cattle tick, Rhipicephalus Boophilus microplus (formerly Boophilus microplus), is the most significant parasite of cattle in Australia and threatens the improvement of cattle production in tropical and subtropical countries worldwide. Some cattle breeds of mainly Bos indicus origin develop a strong resistance to infestation with R. B. microplus, while other breeds of mainly Bos taurus origin will succumb to anaemia and ‘tick worry’ in tick-infested pastures. Resistance to cattle tick infestation is primarily manifest against the larval stage and results in the immature tick failing to make a successful attachment and obtain a meal. It is widely accepted that resistance to tick infestation in cattle is immunologically mediated and involves both cell-mediated and humoral immune responses. Previous research has demonstrated a role for the hypersensitivity response in the rejection of larvae from resistant B. taurus cattle. The immune response to R. B. microplus infestation in B. indicus and B. indicus × B. taurus cattle has not been studied in great detail and this thesis aimed to describe those responses of highly tick-resistant cattle that differ from those of tick-susceptible cattle. A pilot trial was conducted using six tick-resistant Brahman heifers (B. indicus) and six tick-susceptible Holstein-Friesian heifers (B. taurus). The animals were artificially infested for several weeks and blood samples obtained weekly for three weeks during the height of infestation, and on one occasion, skin biopsies obtained. It was found that significant differences existed between the two breeds with respect to the percentage of cellular subsets comprising the peripheral blood mononuclear cell (PBMC) population, cytokine expression by peripheral blood leukocytes (PBL), and levels of tick-specific immunoglobulin G1 (IgG1) antibodies measured in the peripheral circulation. These results in combination with whole genome expression analysis of circulating PBL suggested that the B. indicus cattle had developed a stabilised T cell-mediated response to tick infestation evidenced by their cellular profile and leukocyte cytokine spectrum. The B. taurus cattle demonstrated cellular and gene expression profiles consistent with a sustained innate, inflammatory response to infestation, although high tick-specific IgG1 levels suggested that these animals had also developed a T cell response to infestation. Gene expression analyses using quantitative PCR (qPCR) and whole genome expression platforms indicated that the B. taurus cattle elicited an innate inflammatory response in the skin at the site of larval attachment. This was supported by histological examination of the tick-attachment sites. Conversely, the B. indicus cattle appeared to limit the inflammatory response and instead exhibited increased expression of genes involved in wound healing and the structural integrity of the skin. A second trial was conducted using a group of tick-naïve Santa-Gertrudis heifers to eliminate any innate breed differences that may have confounded the results obtained in the pilot trial. Thirty-five animals were obtained from a tick-free region of Australia; thirty animals were artificially infested weekly for thirteen weeks while five animals remained at a tick-free quarantine property to serve as a control group. Following thirteen weeks of tick infestation the animals in this trial exhibited divergent tick-resistance phenotypes and those carrying the lowest and highest numbers of ticks were classified into tick-resistant (n = 6 animals) and tick-susceptible (n = 6 animals) groups, respectively. The larger number of animals in this study and repeated measurements over the course of the trial enabled a detailed analysis of the effect of tick infestation across all animals, and also within the groups identified as highly resistant or highly susceptible. A cell-mediated response to tick-infestation was detected in all tick-infested animals by twenty-one days after the initial infestation, a response which waned towards the end of the trial when the antibody response became more dominant. Similar to the pilot trial, the tick-susceptible animals developed significantly higher levels of tick-specific IgG1 antibodies compared with the tick-resistant group. Some differences were observed between tick-resistant and tick-susceptible animals with respect to the percentage of cellular subsets comprising the PBMC population. Skin biopsies were taken from these animals prior to initial infestation and then at twenty-one days and ninety-one days after the initial infestation and whole genome expression profiles were produced and analysed. At twenty-one days post infestation both tick-resistant and tick-susceptible animals demonstrated an upregulation of genes involved in leukocyte migration and complement activation, suggestive of an inflammatory response. While this response persisted in the tick-susceptible group at ninety-one days post-infestation, it appeared to lessen in the tick-resistant group. A dominant interleukin-8 (IL-8) response was observed in the skin of the tick-susceptible animals at both time points post-infestation that was not observed in the tick-resistant animals. This thesis demonstrates that tick-susceptible animals respond to infestation with R. B. microplus with an apparently non-protective antibody response, and that tick-resistant animals demonstrate different structural responses in the skin at the tick-attachment site compared with the intense inflammatory response observed in tick-susceptible animals. The results presented here suggest that the extreme susceptibility of pure B. taurus and some B. taurus × B. indicus individuals may be due to their heightened ability to recognise and respond to tick antigens introduced into the host via tick saliva during the blood feeding process. These animals’ increased ability to recognise and respond to these proteins may act to set up a chronic state of inflammation that is beneficial to the tick through increased capillary permeability providing a suitable environment for tick feeding and survival.

O carrapato Rhipicephalus (Boophilus) microplus é responsável por perdas econômicas substanciais na bovinocultura, acarretando o uso intensivo de acaricidas. Problemas com os resíduos químicos presentes na carne e no leite, o custo dos acaricidas e a seleção de populações de carrapato resistentes, têm estimulado o desenvolvimento de métodos de controle alternativos não-químicos. Muito esforço tem sido despendido para o desenvolvimento de uma vacina contra o carrapato, no entanto, seu desenvolvimento depende da identificação de moléculas e caracterização de seus papéis na fisiologia do carrapato. Nesse sentido, entender melhor os processos envolvidos no desenvolvimento embrionário pode ajudar na identificação de alvos para o controle desse ectoparasita. Foi sugerida previamente a participação da Tick Heme-binding Aspartic Proteinase (THAP), uma aspártico-endopeptidase dos ovos do carrapato, na degradação da vitelina. Neste trabalho, nós avaliamos a função fisiológica e as características bioquímicas adicionais dessa proteína. Para identificar os sítios e o perfil de transcrição do gene da THAP, o RNA total foi extraído de intestino, ovário e corpo gorduroso de fêmeas parcialmente e completamente ingurgitadas e de ovos, e analisado por PCR quantitativo. Esta análise revelou que o gene da THAP é transcrito nos três tecidos, porém maior quantidade de mRNA foi detectada no corpo gorduroso e intestino de fêmeas completamente ingurgitadas, onde o processo de vitelogênese já foi iniciado. Nos ovos, a transcrição do gene da THAP não foi detectada. Para investigar a presença da proteína nos tecidos e ovos foi realizado um westen-blot com soro anti-THAP, que revelou a presença, em pequena quantidade, da proteína na hemolinfa, no intestino e no corpo gorduroso. Maior concentração de proteína foi detectada no ovário de fêmeas completamente ingurgitadas e nos ovos durante todo o desenvolvimento embrionário. Também foi observado que a THAP é sintetizada na forma de pró-endopeptidase e depois do início da embriogênese é convertida à forma ativa por autoproteólise. Uma proteína recombinante (rTHAP) foi produzida pela clonagem da região codificadora no vetor de expressão pET43a e expressão em Escherichia coli. Depois da purificação, a rTHAP apresentou atividade enzimática sobre substrato sintético fluorogênico, sendo especificamente inibida por pepstatina A. Para investigar sua participação na degradação de vitelina (VT), VT purificadas de ovos coletados 1, 7 e 12 dias após a postura foram incubadas com a rTHAP em diferentes pH (3,5; 4,0; 4,5; e 5,0). A análise por SDS-PAGE mostrou que a rTHAP é capaz de hidrolisar VT purificada de ovos coletados 7 dias após a postura em pH 3,5 a 37 ºC. Esta atividade é sensível a heme e inibida por pepstatina A. VT purificadas de ovos coletados 1 e 12 dias após a postura não foram hidrolisadas e em outros pH a atividade da rTHAP não foi eficiente. Nossos resultados sugerem que a THAP é sintetizada principalmente em tecidos extra-ovarianos, estocada nos ovários e incorporada nos oócitos como pró-endopeptidase. Durante a embriogênese, a THAP é ativada a enzima na forma madura desenvolvendo papel no processamento da vitelina do carrapato. / Rhipicephalus (Boophilus) microplus is a one-host tick that causes losses to bovine herds, leading intensive use of chemical acaricides. Problems of chemical residues in meat and milk, costs of acaricides, and development of resistance by ticks, have long been recognized and have helped to stimulate interest in tick control by immunological methods. Major efforts have been made to develop vaccines against tick; however, its development still depends on the identification of tick molecules and characterization of their roles in arthropod physiology. In this sense, to understand the processes involved in embryonic development can help in the identification of additional targets to control this ectoparasite. Previously, an aspartic endopeptidase from tick eggs, named THAP (Tick Heme-binding Aspartic Proteinase), was suggested to be involved in vitellin degradation. In this work, we have investigated the physiological role and additional chemical features of this protein. To identify the site and profiles of the THAP transcription, total RNA extracted from midgut, ovary and fat body from partially and fully engorged females and from eggs was analyzed by qRT-PCR. This analysis showed that THAP mRNA was transcripted in these three tissues. However, highest levels of transcriptions were found in fat body and midgut of fully engorged vitellogenic females. In eggs, THAP mRNA transcription was not detected. In order to investigate the presence of THAP protein in the tissues and eggs, an immunoblot analysis was conducted with an anti-nTHAP serum. The THAP protein was detected in the haemolymph, midgut and fat body and, in higher quantity, in the ovary of fully engorged females, and it was present throughout embryo development. The protein is synthesized as a higher molecular mass form (pro-endopeptidase) and after the onset of embryogenesis THAP is converted into an active form by autocatalysis. A recombinant THAP (rTHAP) was produced through cloning in pET43a vector and expression in Escherichia coli. After the purification the rTHAP was active upon fluorogenic substrate in a reaction specifically inhibit by pepstatin A. To investigate rTHAP vitellin-degradation activity, vitellin (VT) purified from 1-, 7- and 12-day-old eggs were incubated with rTHAP in a range of pHs (3.5, 4.0, 4.5 and 5.0). SDS-PAGE analysis showed that rTHAP is able to hydrolyze VT from 7-day-old eggs in pH 3.5 at 37ºC in a reaction that is heme-sensitive and inhibited by pepstatin A. Vitellins from eggs collected on the 1st and 12th days after oviposition were not hydrolyzed and in other pHs rTHAP activity was not efficient. These results suggest that THAP is synthesized in ovary and extra-ovarian site, stocked in ovary and incorporated by vitellogenic oocytes with a pro-endopeptidase. During embryogenesis, THAP was actived to the mature enzyme and play a role in tick vitellin processing.