No Brasil, carrapatos do gênero Rhipicephalus são representados pelas espécies Rhipicephalus sanguineuss.l. (Latreille) e Rhipicephalus (Boophilus) microplus (Canestrini), sendo que a primeira espécie possui especificidade com cães domésticos e a segunda com bovinos. Para o cão, R. sanguineus s.l. é o principal vetor de agentes causadores da babesiose canina e da erliquiose, enquanto que nos bovinos, R. microplus é responsável, principalmente, pela transmissão de agentes causadores da babesiose e anaplasmose. Alguns dos patógenos causadores dessas doenças são difíceis de serem cultivados in vitro, e não crescem em meios artificiais, especialmente Anaplasma spp. Assim, muitas linhagens celulares de carrapatos foram desenvolvidas nos últimos 40 anos e têm sido amplamente utilizadas para isolamento e propagação de microrganismos patogênicos. Cultivos celulares obtidos de células embrionárias de carrapatos oferecem um sistema de vetor in vitro, que é útil principalmente para estudos de agentes intracelulares. Dessa forma, a obtenção de culturas de células embrionárias de R. sanguineus (origens tropical e temperada) e de R. (B.) microplus, bem como, sua infecção com Ehrlichia canis e Anaplasma marginale, respectivamente, são objetivos do presente estudo. Os cultivos celulares dessas espécies de carrapatos foram preparados com massas de ovos de diferentes idades e mantidos à 30 °C em meio de cultura L15-B, suplementado com 10% de Triptose Fosfato e 20% de Soro Fetal Bovino. Subcultivos foram realizados após a formação da monocamada celular confluente. As identidades celulares foram confirmadas pela PCR e sequenciamento, utilizando um fragmento do gene mitocondrial 16S rDNA. As sequências das culturas de células de R. sanguineus (tropical e temperada) e de R. microplus foram depositadas no GenBank. Essas culturas de células foram infectadas com E. canis e A. marginale (cepas Jaboticabal), respectivamente, e mantidas em meio L-15B (Vitrocell) suplementado com Soro Fetal Bovino enriquecido com ferro (Hyclone) nas concentrações de 2% e 5%. As células infectadas foram mantidas em propagações sucessivas até a terceira passagem, para novas culturas de células não infectadas, sendo então criopreservadas. O DNA extraído das células infectadas e não infectadas de R. sanguineus (de ambas as origens) e de R. microplus, foi analisado pela PCR quantitativa em tempo real (qPCR), utilizando os genes E. canis-dsb e msp1β, respectivamente. Propagações de células de carrapatos infectadas para as novas culturas de R. sanguineus (origem tropical) e R. (B) microplus, foram bem sucedidas. Por outro lado, as células de R. sanguineus (origem temperada) não se tornaram infectadas no presente estudo. / In Brazil, ticks of the genus Rhipicephalus are represented by the species Rhipicephalus sanguineus s.l. (Latreille) and Rhipicephalus (Boophilus) microplus (Canestrini), being that the first one has specificity with domestic dogs and the second with cattle. For dogs, R. sanguineus s.l. is the main vector of causative agents of canine babesiosis and ehrlichiosis, whereas in cattle, R. microplus is responsible, mainly, for the transmission of agents that cause babesiosis and anaplasmosis. Some of the pathogens that cause these diseases are difficult to grow in vitro, and do not grow on artificial media, especially Anaplasma spp. Therefore, many tick cell lines were developed in the last 40 years and have been used for the isolation and propagation of pathogenic microorganisms. Cell cultures obtained from embryonic tick cells offer an in vitro vector system, which is mainly useful for studies of intracellular agents. The aim of the present study was to obtain cultures of R. sanguineus (tropical and temperate lineages) and R. microplus embryonic cells and their infection with Ehrlichia canis and Anaplasma marginale. Cell cultures from the tick species were prepared with egg masses of different ages and kept at 30 ° C in L15-B culture medium supplemented with 10% Tryptose Broth and 20% Fetal Bovine Serum. Subcultures were done after confluent cell monolayer formation. Cellular identities were confirmed by PCR and sequencing using a 16S rDNA mitochondrial gene fragment. Sequences of R. sanguineus (tropical and temperate) and R. microplus cell cultures were deposited on GenBank. These cell cultures were infected with E. canis and A. marginale (Jaboticabal strains), respectively, and maintained in L-15B medium and Fetal Bovine Serum with iron (Hyclone) supplemented at 2% and 5% concentrations. The infected cells were maintained in successive propagations until the third passage, to new cultures of uninfected cells, and then were cryopreserved. DNA extracted from infected and uninfected R. sanguineus (both origin) and R. microplus cells was analyzed by quantitative real-time PCR (qPCR) using the E. canis-dsb and msp1β genes, respectively. Propagations of infected tick cells to the new cultures of R. sanguineus (tropical origin) and R. microplus, were successful. On the other hand, the R. sanguineus cells (temperate origin) did become infected in the present study.

O carrapato bovino Rhipicephalus (Boophilus) microplus é o principal vetor da riquétsia Anaplasma marginale, o agente etiológico da anaplasmose, uma doença que acomete os rebanhos e causa sérios prejuízos econômicos à pecuária no Brasil. Estabelecemos em nosso laboratório o cultivo da linhagem de células BME26 que são originárias do R. (B.) microplus e também a infecção dessas células por A. marginale, um patógeno que é naturalmente transmitido pelo carrapato. Detectamos que a expressão gênica da defensina e da ixodidina nas células é aumentada frente à infecção por A. marginale, embora nenhuma alteração da expressão gênica da microplusina foi constatada. As células foram expostas a microorganismos inativados por calor e LPS, sendo que a expressão gênica da microplusina é aumentada frente a todos os estímulos. Na exposição das células BME26 com a bactéria Microccocus luteus, a expressão gênica da defensina e da ixodidina não foi alterada e no estimulo com leveduras a expressão gênica da ixodidina foi reprimida. Frente à infecção por A. marginale detectamos, aumento expressão da defensina e ixodidina. Os genes da microplusina e defensina foram silenciadas por RNAi em células infectadas por A. marginale, mas não houve alteração no número de riquétsias / The cattle tick Rhipicephalus (Boophilus) microplus is the main vector of the rickettsia Anaplasma marginale, the etiological agent of anaplasmosis, a disease that affects cattle and causes serious economic losses to the Brazilian cattle industry. We established in our laboratory the embryonic cell culture line BME26 from R. (B.) microplus and infection by A. marginale, a pathogen naturally transmitted by R. (B.) microplus. We verified that defensin and ixodidin gene expression increased in these cells after an infection by A. marginale and no alteration in microplusin gene expression was detected. The BME26 cells were exposed to heat-inactivated microorganims or to LPS, microplusin gene expression increased after all stimuli. After exposure of BME26 cells to Micrococcus luteus, expression levels of defensin and ixodidin did not change and ixodidin gene expression reduced after exposure of these cells to yeast. In the infection by A. marginale we detected defensin and ixodidin gene expression. Also, microplusin and defensin genes were silenced by RNA interference (RNAi) in A. marginale-infected BME26 cells, but we did not observe alteration in the number of MSP4 rickettsias

This research was aimed to evaluate in vivo effects of citronella oil on cattle ectoparasites. Two experiments with different levels of citronella oil (I) and the relationships with phytotherapic and the synthetic acaricide (II) were evaluated. In experiment I, twenty Holstein cows were allocated in four groups of five animals. The treatments were: negative control, amitraz at 0.025% and citronella oil at 3 and 4%. Engorged ticks and flies (horn fly, stably fly and domestic fly) were evaluated on animals before (mean of days -3, -2, -1) and at 3; 7 and 14 days after treatment. The efficacy of control ticks was 89.2; 36.5 and 35.8% for amitraz and citronella oil at 3 end 4% respectively, on 14th day post-treatment. Lower control of flies was observed for phytotherapic treatments. In experiment II, fifteen Holstein cows were allocated in three groups of five animals. The treatments were: negative control, amitraz at 0.025% and citronella oil at 4%. Engorged ticks and flies were evaluated on animals before (mean of days -3, -2, -1) and at 7; 14; 21 and 28 days after treatment. In 28 days there was need to reapply the amitraz to control engorged ticks. The relationship among the number of application was the 1:2.5 for amitraz and citronella oil, respectively. The efficacy in control ticks were 71.8 and 30.9% for amitraz and citronella oil at 4% respectively. Lower control of flies was observed for phytotherapic treatment. / O objetivo desta pesquisa foi avaliar in vivo diferentes níveis de óleo de citronela em ectoparasitas de bovinos. Foram conduzidos dois experimentos com diferentes níveis de óleo de citronela (I) e a relação de banho entre o produto fitoterápico e um acaricida sintético (II). No experimento I, foram utilizadas 20 vacas da raça Holandês, distribuídas em quatro grupos de cinco animais cada um. Os tratamentos foram constituídos pelo controle negativo, amitraz a 0,025%, óleo de citronela a 3 e 4%. Para avaliação foram contadas fêmeas ingurgitadas de carrapato e moscas (mosca-dos-chifres, mosca-dos-estábulos e mosca doméstica) antes (média dos dias -3, -2, -1) e após a aplicação dos produtos nos dias 3; 7 e 14. A eficácia no controle do carrapato foi de 89,2; 36,5 e 35,8% para o amitraz e óleo de citronela a 3 e 4%, respectivamente. Verificou-se baixo controle de moscas nos tratamentos constituídos pelo fitoterápico. No experimento II, foram utilizadas 15 vacas da raça Holandês, distribuídas em três grupos de cinco animais cada um. Os tratamentos foram constituídos pelo controle negativo, amitraz a 0,025% e óleo de citronela a 4%. Para avaliação foram contadas fêmeas ingurgitadas de carrapato e moscas antes (média dos dias -3, -2, -1) e após a aplicação dos produtos nos dias 7; 14; 21 e 28. Aos 28 dias, houve necessidade de se reaplicar o amitraz para controlar a infestação com carrapato. A relação entre o número de aplicações foi de 1:2,5 para amitraz e citronela, respectivamente. A eficácia no controle do carrapato foi de 71,8 e 30,9% para o amitraz e óleo de citronela a 4%, respectivamente na média pós-tratamento. Verificou-se baixo controle de moscas no tratamento constituído pelo fitoterápico.

Submitted by Renata Lopes (renatasil82@gmail.com) on 2017-05-17T19:13:24Z No. of bitstreams: 1 felipemartinsferreira.pdf: 5165550 bytes, checksum: dffda206ccbfc77d5aa53ac793323b11 (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2017-05-18T11:37:40Z (GMT) No. of bitstreams: 1 felipemartinsferreira.pdf: 5165550 bytes, checksum: dffda206ccbfc77d5aa53ac793323b11 (MD5) / Made available in DSpace on 2017-05-18T11:37:40Z (GMT). No. of bitstreams: 1 felipemartinsferreira.pdf: 5165550 bytes, checksum: dffda206ccbfc77d5aa53ac793323b11 (MD5) Previous issue date: 2016-07-20 / Os carrapatos são importantes vetores de doenças em animais e humanos. O carrapato dos bovinos, Rhipicephalus (Boophilus) microplus (Canestrini, 1887) é responsável por sérios prejuízos à pecuária brasileira considerando-se os custos do controle, perda de receita devido a menor produção de leite e carne, danos ao couro, além da transmissão de doenças. O uso de plantas medicinais e de seus derivados com ação carrapaticida é considerado um excelente recurso frente a formação de resistência aos produtos químicos. Diante da necessidade de alternativas mais eficazes e com menores impactos ambientais o objetivo desse estudo foi avaliar a ação carrapaticida in vitro do eugenol, do óleo essencial e do hidrolato de Syzygium aromaticum (cravo-da-índia). Adicionalmente, também foi investigada a atividade carrapaticida do óleo essencial e do eugenol quando incorporados em uma formulação. O óleo essencial e o hidrolato de cravo-da-índia foram obtidos pelo processo de hidrodestilação dos botões florais secos da planta. A caracterização química do óleo essencial de cravo-da-índia por CG/EM revelou a presença dos componentes majoritários eugenol (83,97%), cariofileno (3,04%), humuleno (0,42%) e acetato de eugenol (12,58%). Os carrapatos provenientes de diversas regiões geográficas foram tratados com os compostos em estudo em diferentes etapas de seu ciclo. No teste de pacote de larvas foi avaliada a mortalidade com concentrações de eugenol, óleo essencial e hidrolato que variaram de 0,1 à 2,0%. O índice de produção de ovos, a eclosão larval e a eficácia carrapaticida foram avaliados através do teste de imersão de fêmeas ingurgitadas, em que foram utilizadas concentrações de eugenol e óleo essencial de 1,25 a 10%. O eugenol e o óleo essencial promoveram 100% de mortalidade em larvas a partir das concentrações de 0,25 e 0,5%, respectivamente. O hidrolato não apresentou atividade carrapaticida nas concentrações testadas. No teste de imersão de fêmeas o eugenol e o óleo essencial apresentaram resultado de eficácia superior a 95% a partir da concentração de 5,0%, inibindo completamente a eclosão larval na concentração de 10%. O uso do eugenol, substância purificada (99-100%) não proporcionou superioridade significativa nas eficácias de tratamento em relação ao óleo essencial de cravo, o que reforça o potencial do uso do óleo essencial de cravo como carrapaticida, uma vez que o mesmo é uma alternativa de fácil acesso, baixo custo e com alto rendimento no processo extrativo. A formulação desenvolvida não promoveu melhores eficácias de tratamento quando comparada a solução etanólica, o que sugere que novas pesquisas deverão ser realizadas sob a perspectiva de testar o óleo essencial de cravo em outras formulações que possam melhorar sua atividade. Além disso, testes in vivo são recomendados para validação da eficiência do ativo em condições de campo. / Ticks are important vectors of diseases in animals and humans. The cattle tick Rhipicephalus (Boophilus) microplus (Canestrini, 1887) has caused serious harm to livestock raising in Brazil, considering the costs of controlling it, revenue loss as a result of a smaller production of milk and meat, and leather damage, in addition to transmitting diseases. The use of medicinal plants and their derivatives with acaricide action is considered an alternative against the formation of chemicals resistance. Due to the need for more efficient alternatives with less environmental impact, this study aims to evaluate the acaricide action in vitro of eugenol, essential oil and hydrolate of Syzygium aromaticum (clove). Additionally, it has also investigated the acaricide activity of the essential oil and eugenol when incorporated into a formulation. The extraction of the essential oil and hydrolate was performed by steam distillation of dried flower buds of the plant. The chemical characterization of clove essential oil by GC/MS revealed the presence of the major components eugenol (83.97%), caryophyllene (3.04%), humulene (0.42%), and eugenol acetate (12.58%). The ticks from different geographical areas were treated with the test compounds in different stages of the tick cycle. In the larval packet test, mortality was assessed at concentrations of eugenol, essential oil and hydrolate ranging from 0.1 to 2.0%. The eggs production levels, the larvae hatching, and their efficiency on ticks were assessed using the adult immersion test, in which eugenol and essential oil were used at concentrations of 1.25 to 10%. The eugenol and the essential oil caused 100% mortality of larvae, starting at 0.25 and 0.5%, respectively. The test with hydrolate showed no activity at the concentrations tested. In the adult immersion test, the eugenol and the essential oil showed results greater than 95% efficacy at concentrations of 5.0%, completely inhibiting the larvae hatching at 10%. The use of eugenol, purified substance (99-100%), gave no significant superiority in efficacy of treatment with the clove essential oil, which strengthens the potential use of clove essential as an acaricide, being it a low cost and accessible alternative, with high yield in the extraction process. The formulation developed did not promote better efficacies of treatment when compared to ethanol solutions, which suggests that further research should be conducted from the perspective of testing the essential oil of cloves in other formulations that can improve their activity. Furthermore, in vivo tests are recommended for an active efficiency in field conditions validation.

Made available in DSpace on 2014-08-20T14:38:00Z (GMT). No. of bitstreams: 1 dissertacao_anibal_torres.pdf: 438835 bytes, checksum: d43cf21b0af9852e79231f87a87e06ea (MD5) Previous issue date: 2010-02-22 / The equine theileriosis, caused by Theileria equi, is an endemic disease of south of Rio Grande do Sul Brazil. There are signs that tick Rhipicephalus boophilus microplus still transmit that disease and in few time, some studies had experimental demonstrated it. The purpose of the first study of this paper was to demonstrate the tick infestation and the role of it in the transmission of the disease. In equines with contact directly with cattle, the serum incidence of the disease was 81,8% and in this group, 31,8% of this horses had tick infestation. In the equines that didn t have contact with cattle, the incidence of the disease was 12% and no tick was found in this horses. The most efficient drug in the treatment of the disease is imidocarb dipropionate. Many studies had demonstrated different doses and protocols of treatment of the disease and the toxicity of it was obvious. No study had show the sterile of T. equi with this drug, but it s a way to treat the acute disease and to control the chronic theileriosis. The acute form of the disease is seemed with fever, icteric and death. The chronic kind of it is seemed with covered coat, hyporexia, low performance in athlete horses and loss of weight. The goal of the second study of this paper was to show the toxicity and the metabolic effects of imidocarb dipropionate drug into two protocols of treatment ( two groups, 2 and 4mg/kg) in serum positive horses to theileriosis. It was seemed through hepatic and renal profile that 2mg/kg of the drug have low toxicity. Horses infected with T. equi keep with the disease for the rest of your lives, and the drug control with imidocarb dipropionate is necessary. The goal of the third study of this paper was to demonstrated, through immunologic test and clinic examination that 2mg/kg of imidocarb dipropionate monthly can control the chronic disease even in stress situations. One group received monthly, through 6 mouths, 2mg/kg of imidocarb dipropionate and the other group, once, in the beginning of it, 4mg/kg. The conclusion of this paper is that the incidence of equine theileriosis have a directly relation with the catle contact because it cause the tick Rhipicephalus Boophilus mircoplus infestation. Mensal dosis with 2mg/kg of imidocarb dipropionate is efficient in the disease control and it had demonstrated that it is not toxic for equine. / A theileriose equina, causada pelo hemoprotozoário Theileria equi, é uma doença endêmica na região da campanha do Rio Grande do Sul. Há indícios de que o carrapato Rhipicephalus Boophilus microplus transmita também a theileriose equina e, há pouco tempo, estudos demonstraram experimentalmente esta transmissão. O objetivo do primeiro trabalho foi demonstrar a infestação de carrapatos Rhipicephalus Boophilus microplus e o papel importante deste parasita na transmissão de theileriose equina. Em cavalos com contato direto com bovinos, a incidência sorológica da doença foi de 81,8%, sendo que em 31,8% destes animais se encontrou carrapatos Rhipicephalus Boophilus microplus. Nos equinos sem contato com bovinos, a incidência sorológica foi de 12% e não se encontrou carrapatos. Diversos estudos têm demonstrado a eficiência no tratamento da doença com dipropionato de imidocarb em diferentes doses, porém a toxicidade deste fármaco se manifesta em alguns animais. A forma aguda é caracterizada por febre, icterícia e morte. A forma crônica é descrita por pêlo arrepiado, hiporexia, queda no desempenho em animais atletas e perda de peso. O objetivo do segundo trabalho foi demonstrar a toxicidade e os efeitos metabólicos do dipropionato de imidocarb em duas doses terapêuticas (2 e 4mg/kg). Foi sugerido, através do perfil hepático e renal, após a administração da droga, que 2mg/kg deste fármaco tem uma toxicidade leve e temporária no período de metabolização da droga. Uma vez infectados com a T. equi, os equinos permanecem a vida toda positivos para a doença. Com isso o controle da enfermidade depende da utilização do dipropionato de imidocarb. O objetivo do terceiro trabalho foi demonstrar, através do teste de imunofluorescência indireta e exame clínico, que doses mensais de dipropionato de imidocarb á 2 mg/kg são eficazes para se manter a parasitemia e a clínica da doença controladas, mesmo que os animais sejam submetidos á situações de estresse. Um grupo recebeu mensalmente, por seis meses, 2mg/kg de dipropionato de imidocarb e o outro grupo, 4mg/kg apenas uma vez no primeiro mês. A conclusão desta dissertação é que a incidência da theileriose equina tem relação direta com a convivência com bovinos por facilitar a infestação destes com o carrapato Rhipicephalus Boophilus microplus; e que, doses mensais de dipropionato de imidocarb, à 2mg/kg, são eficazes no controle da parasitemia da doença mesmo em situações de estresse e não se demonstram tòxicas para os equinos.

Made available in DSpace on 2014-08-20T14:38:04Z (GMT). No. of bitstreams: 1 tese_felipe_geraldo_pappen.pdf: 1221798 bytes, checksum: 72f7cef0d47255d268dc994513126704 (MD5) Previous issue date: 2011-09-21 / Practices of Rhipicephalus (Boophilus) microplus control are problematic in many livestock production systems and have worldwide increased. In the south region of Rio Grande do Sul State, Brazil, where beef cattle are breed, dip baths are the main option for acaricide treatments. Thus, the aim of this study consist in: (i) to know the epidemiological aspects of dip baths use for R. (B.) microplus control in south of Brazil, (ii) to evaluate chemical sensibility of R. (B.) microplus in beef farms with cattle dip baths, (iii) to evaluate dip baths structure and maintenance conditions, and (iiii) to evaluate tick control handlings procedures. An epidemiologic inquiry was applied to 110 beef farmers holding plunge cattle dips, which were located in eight tows in the south region. The obtained results showed that dip baths were unable on 27.3% (30/110) of the properties,mainly due to the presence of resistance to most of the acaricide classes (40% - 12/30). Dip baths have been active in 72.7% (80/110) and amitraz is the most used acaricide in 82.5% (66/80) of these farms, followed by associations of pyrethroids and organophosphates in 16.2% (13/80), and nonassociated pyrethroids in 1.3% (1/80) of properties. Suitable number of engorged female ticks to laboratory sensibility test were obtained in 17.5% (14/80) of farms, demonstrating that 42.9% (6/14) of tick populations were sensible to amitraz, while 85.7% (12/14) were susceptible to associations, and none was sensible to pyrethroids. Moreover, in vitro tests revealed that dip baths solutions were only efficient in 35.7% (5/14) of them. Although the dip baths presents a mean age of 35 years, most farmers have no claims of physical infrastructure issues. The main criterion for the decision of the immersion baths application is the visualization of the immature ticks on cattle in 66% (53/80) of the properties and five or more applications per year were performed in 65% (52/80) of them. Moreover, the simultaneous use of other acaricides on injectable forms and pour-on, and the extension of the parasitism period to the winter months suggested that tick control in the region is reason for concern. It was also concluded that pyrethroids have been replaced by amitraz in last years, and nowdays this acaricide is being replaced by associations of pyrethroids with organophosphates, a viable alternative for most of the farms according to the in vitro tests. / O controle do carrapato Rhipicephalus (Boophilus) microplus é problemático em vários sistemas de criação, estando disseminados os casos de resistência por todo o mundo. A situação também é preocupante na região sul do Rio Grande do Sul, que por sua vez utiliza o banheiro de imersão como principal método de aplicação de acaricidas em propriedades de criação de bovinos de corte. Assim, o presente trabalho teve os seguintes objetivos: (i) conhecer os aspectos relacionados à utilização dos banheiros de imersão para o controle do carrapato R. (B.) microplus; (ii) avaliar a sensibilidade de R. (B.) microplus destas propriedades; (iii) avaliar as características e condições de estrutura física dos banheiros de imersão; e (iiii) avaliar itens de manejo relativos ao controle do carrapato. Para tanto, um questionário epidemiológico foi aplicado em 110 propriedades de criação de bovinos de corte com banheiro de imersão, distribuídos em oito municípios da região de estudo. Constatou-se que em 27,3% (30/110) delas, o banheiro carrapaticida encontra-se desativado, principalmente em virtude do surgimento de resistência aos princípios ativos disponíveis (40% - 12/30). Naquelas em que o banheiro de imersão está carregado (72,7% - 80/110), o amitraz é o ingrediente ativo mais utilizado (82,5% - 66/80), seguido das associações de piretróides com organofosforados (16,2% - 13/80) e dos piretróides não associados (1,3% - 1/80). Obteve-se número suficiente de teleóginas para a avaliação de sensibilidade em laboratório em 17,5% (14/80) das propriedades, constatando-se que 42,9% (6/14) das populações eram sensíveis ao amitraz, 85,7% (12/14) às associações de piretróides com organofosforados, e, nenhuma aos piretróides não associados. Os testes in vitro também revelaram que as caldas utilizadas eram eficazes em apenas 35,7% (5/14) dos casos. Embora os banheiros de imersão da região possuam, em média, 35 anos de existência, e alguns problemas conservação tenham sido constatados, a maioria dos proprietários alega possuir estrutura física adequada. O critério para a realização dos banhos é a visualização de formas imaturas do carrapato sobre o bovino em 66% (53/80) das propriedades e são realizadas cinco ou mais aplicações em 65% (52/80) delas. Além disso, a utilização simultânea de outros ingredientes ativos nas formas injetável e pour-on, e a extensão do período de parasitismo aos meses de inverno sugerem que a situação do controle do carrapato na região está se agravando. Também conclui-se que os piretróides foram ao longo dos últimos anos substituídos pelo amitraz, que por sua vez vem dando lugar às associações de piretróides com organosfosforados, uma alternativa viável para a maioria das propriedades segundo os testes in vitro.

Orientador: Sergio Daishi Sasaki / Dissertação (mestrado) - Universidade Federal do ABC, Programa de Pós-Graduação em Biossistemas, 2013

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.

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.