Orientador: Adjair Antonio do Nascimento / Banca: Gilson Pereira de Oliveira / Banca: Júlio Mendes / Resumo: O presente trabalho procurou verificar a possível interferência do pH do diluidor na eficiência de ixodicidas comerciais. Para tanto, utilizou-se amitraz, clorpirifós, cipermetrina, mais um grupo controle, para verificar os possíveis efeitos de soluções tampão com pH 3, 4, 5, 6, 7, 8, 9, 10 e 11, como diluidores dos ixodicidas, sobre as variáveis: peso médio da oviposição (PMO), relação PMOxPT (peso das teleóginas), porcentagem de eclosão (PE), eficiência reprodutiva (ER) e eficácia da calda (EC). Teleóginas de R. (B.) microplus, obtidas de bovinos infestados com cepa de campo, foram submetidas à biocarrapaticidogramas, uma hora e 24 horas após preparo (HAP) das caldas. O pH do diluente pouco influenciou na eficácia do amitraz nos parâmetros avaliados, embora as linhas de tendência dos resultados para ER e EC tenham mostrado menor ação do ixodicida, à medida que o pH do diluidor aumentava, tanto para uma HAP como para 24 HAP. Para o clorpirifós, os resultados obtidos mostraram diferenças significativas no PMO, PMOxPT e PE nos resultados de uma hora. A ER (uma HAP) e EC (24 HAP) foram influenciadas negativamente pelo pH quando este se elevava. As linhas de tendência dos resultados para ER e EC mostraram diminuição da ação à medida que o pH do diluidor aumentava, tanto para uma hora como para 24 horas. A cipermetrina sofreu pouca influência do pH. Entretanto, a linha de tendência dos resultados para EC foi influenciada pela elevação do pH, quando usada 24 HAP. / Abstract: The objective of the present research is to check, with commercial ixodicides, the possible interference of the pH of the thinner. For this amitraz, chlorpyrifos, cypermethrin, plus a control group, was used to verify the possible effects of buffer solutions with pH 3, 4, 5, 6, 7, 8, 9, 10 and 11, as thinners of the ixodicides, on the following: medium weight of egg mass (PMO), relationship PMOxPT (weight of the teleogines), percentage of larvae hatch (PE), estimated reproduction (ER) and % control (EC). Teleogines of R. (B.) microplus, obtained from cattle infested with field's strains have been submitted to the in vitro immersion tests, one hour and 24 hours after the solution has been prepared (HAP). The pH of the thinners made little difference on the amitraz on the parameters assessed and, statistically, did not show any significant differences, although the tendency of the results for ER and EC showed less action of ixodicide, as the pH of the thinner increased, as much for one HAP as for 24 HAP. As for chlorpyrifos, the results obtained, statistically showed significant differences on the PMO, PMOxPT and PE in the results of one hour. The ER (one HAP) and EC (24 HAP), were negatively affected by pH when this was augmented. The tendency of the results for ER and EC showed a diminution from the action, as the pH from the thinner augmented, as much for one HAP as for 24 HAP. The cypermetrin suffered less influence from pH. However, the tendency of the results for EC were affected by the elevation of pH, when used 24 HAP. / Mestre

Made available in DSpace on 2016-04-28T20:15:27Z (GMT). No. of bitstreams: 1 2007-Isabele da Costa Angelo.pdf: 1629986 bytes, checksum: 663b35706e830a57e185c3e0a68096c9 (MD5) Previous issue date: 2007-02-26 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / Boophilus microplus is an ectoparasite which causes great economic losses around the world. The exclusive use of acaricides and the inadequate management have conducted the development of resistance in ticks populations, environmental and food contamination by acaricides and their residues. The use of entomopathogenic fungi to arthropods control has shown interesting responses. Several studies have proved the pathogenicity of fungi toward various ticks species. The aim of the present study was to evaluate the effect of Isaria farinosa, I. fumosorosea, Paecilomyces lilacinus and Lecanicillium lecanii toward engorged females, eggs and larvae of B. microplus tick. The isolates were cultured on malt extract medium at 25 ? 1?C and 80% of relative humidity for 15 days. Conidial suspensions were prepared in Tween 80 water solution (0.1% v/v). There were 4 treatment groups according to the following conidial concentrations: 105, 106, 107 and 108 conidia mL-1. The control group was made up of water and Tween 80 only (0.1% v/v). Treatment was based on immersion of the specimen in 1 mL of the conidial suspension. Each treatment group was made up of 10 repetitions. Changes in biology of engorged females, eggs viability and larvae mortality, were observed every 5 days up to the 20th day after treatment. The results have shown that L. lecanii changed biological parameters in engorged females through the reduction in posture period, nutritional rate, eggs production rate, and increase of incubation period. Isaria farinosa has shown reduction in posture period and in nutritional rate. Paecilomyces lilacinus was the unique isolate able to reduce the eclosion period of larvae from infected females. Isaria fumosorosea has reduced the nutritional rate and was the unique isolate that changed significantly in eclosion period when eggs were infected. Conidial concentrations reduced reproduction capacity of engorged females. The isolates of I. fumosorosea and L. lecanii have presented major potential to control B. microplus engorged females. All tested isolates have shown pathogenicity toward unfed larvae of B. microplus after in vitro infection. / Boophilus microplus ? um ectoparasito que causa grandes perdas na pecu?ria mundial. A utiliza??o exclusiva de produtos qu?micos no controle de carrapatos, associada ao manejo inadequado tem conduzido o desenvolvimento de popula??es de carrapatos resistentes, e a contamina??o dos produtos de origem animal e do ambiente pelos seus res?duos. O uso de fungos entomopatog?nicos no controle de artr?podes tem se tornado uma abordagem cada vez mais atrativa. In?meros trabalhos comprovam experimentalmente a patogenicidade dos fungos sobre diversas esp?cies de carrapatos. O objetivo do presente trabalho foi avaliar o efeito in vitro de Isaria farinosa, I. fumosorosea, Paecilomyces lilacinus e Lecanicillium lecanii sobre f?meas ingurgitadas, ovos e larvas de B. microplus. Os fungos foram repicados em meio de cultura extrato de malte, e mantidos em c?mara climatizada sob temperatura de 25?C ? 1 e umidade relativa de 80% por quinze dias. Suspens?es conidiais foram preparadas a partir do crescimento do fungo, cujos con?dios foram adicionados a solu??o de ?gua destilada e Tween 0,1%. As concentra??es 105, 106, 107 e 108 con?dios mL-1 formaram os grupos tratamento, juntamente com o grupo controle, constitu?do por ?gua destilada est?ril e Tween 0,1%. O tratamento constituiu-se de um mililitro da concentra??o conidial testada, e cada grupo foi formado por 10 repeti??es. Os par?metros de avalia??o observados para demonstrar o efeito dos fungos sobre o carrapato foram as altera??es biol?gicas de f?meas ingurgitadas, viabilidade de ovos tratados e percentual de mortalidade de larvas, acompanhado a cada cinco dias at? o 20? dia ap?s infec??o. Os resultados mostraram que L. lecanii causou altera??es nos par?metros biol?gicos de f?meas ingurgitadas, diminuindo o per?odo de postura, o ?ndice nutricional, o ?ndice de produ??o de ovos, e aumentando o per?odo de incuba??o. Isaria farinosa mostrou redu??o no per?odo da postura e no ?ndice nutricional de f?meas ingurgitadas. P. lilacinus foi o ?nico fungo capaz de reduzir o per?odo de eclos?o das larvas provenientes da infec??o de f?meas ingurgitadas. I. fumosorosea reduziu o ?ndice nutricional e foi o ?nico isolado que causou altera??o significativa no per?odo de eclos?o de larvas provenientes da infec??o de ovos. Diferentes concentra??es dos entomomopat?genos reduziram o potencial reprodutivo das f?meas ingurgitadas dos grupos tratados. Os isolados de I. fumosorosea e L. lecanii apresentaram maior potencial de controle para f?meas ingurgitadas de B. microplus. Todos os isolados testados mostraram patogenicidade para larvas n?o alimentadas de B. microplus ap?s infec??o in vitro.

Made available in DSpace on 2016-04-28T20:16:21Z (GMT). No. of bitstreams: 1 2007-Everton Kort Kamp Fernandes.pdf: 2844359 bytes, checksum: a56e9f806dc8f1f0e7cc6b4641b4a78c (MD5) Previous issue date: 2007-03-30 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico / Beauveria bassiana is an entomopathogenic fungus used worldwide as a biological control agent of pest arthropods. The present study aimed at characterizing several isolates of B. bassiana and selects those with major potential to control ticks population and other arthropods under natural conditions. Thus, 53 isolates of B. bassiana and 7 isolates including 5 other Beauveria spp. and 1 Engyodontium albus (=Beauveria alba) were investigated. These isolates were originally from different regions of Brazil and other nations, and different hosts or substrate. Initially, all the isolates were morphologically analyzed and investigated about the potential of conidial production. Afterwards, experiments were conducted to investigate the genetic diversity of Brazilian isolates of B. bassiana, to evaluate the spatial distribution of genotypes, and to understand the genetic relationship among isolates of different species of Beauveria. Thus, all isolates were analyzed by isozymes electrophoresis, AFLP-PCR and sequencing of ITS1 and ITS2 regions. The results have permitted the taxonomic identification of isolates and the selection of those with major potential to conidial production. The results have also showed the detection of high genetic variability among B. bassiana isolates, indicating that this fungus represents a species aggregate. Furthermore, the genetic distance among isolates was directly related to the geographic distances. In addition, the isolates were evaluated to UV-B tolerance, heat tolerance, and cold activity. Heat and cold are two abiotic factors of the environment that restrict the use of entomopathogenic fungi as agents for biological control of insects. In addition, solar radiation, particularly the UV-B wavelength, is a major environmental factor that can negatively affect this entomopathogenic fungus in the field. High variability in UV-B tolerance was detected among the isolates after 2 h of radiation exposure. Conidial relative germination ranged from 0% to almost 80%, and a delayed germination was detected. Moreover, isolates of B. bassiana originating from lower latitudes tended to have lower UV-B tolerance than isolates from higher latitudes. A high variability in thermotolerance was also observed among the isolates after 2 h of heat exposure at 45 ?C, ranging from low, to medium, to high viability. The thermal death point of most resistant isolates was between 44 ?C and 45 ?C after 6 h of heat exposure. At cold temperatures most of the isolates grew at 5 ?C. An attempt to correlate the latitude of origin with thermotolerance or cold activity indicated that isolates from higher latitudes were more cold-active than isolates from nearer the equator. The present study also evaluated the virulence of the isolates toward Boophilus microplus larvae, and virulence ranged from very low to very high. It was observed that different populations of B. microplus may present different susceptibility to B. bassiana infection. Therefore, the present study has selected isolates of this entomopathogenic fungus with major effective potential to control B. microplus and other pest arthropods under environmental conditions. / Beauveria bassiana ? um fungo entomopatog?nico utilizado em v?rias partes do mundo como agente de controle biol?gico de artr?podes. Baseado nisso, o presente estudo buscou caracterizar diversos isolados de B. bassiana e selecionar aqueles com maior potencial para controlar popula??es de carrapatos e outros artr?podes em condi??es naturais. Neste sentido, 53 isolados de B. bassiana e mais sete isolados incluindo cinco esp?cies de Beauveria spp. e uma de Engyodontium albus (=Beauveria alba) foram estudadas. Esses isolados foram oriundos de v?rias regi?es geogr?ficas brasileiras e estrangeiras, e de diferentes hospedeiros ou substratos. Os isolados foram inicialmente avaliados morfologicamente e quanto ao potencial de produ??o de con?dios. Em seguida, buscou-se investigar a diversidade gen?tica dos isolados brasileiros de B. bassiana, avaliar a distribui??o espacial de gen?tipos, e conhecer a rela??o gen?tica entre os isolados das diferentes esp?cies. Para esta finalidade os isolados foram submetidos as t?cnicas de eletroforese de isoenzimas, AFLP-PCR e seq?enciamento de regi?es ITS1 e ITS2. Os resultados permitiram a confirma??o taxon?mica dos isolados e a sele??o daqueles com maior capacidade de produ??o de con?dios. Permitiram tamb?m detectar grande variabilidade gen?tica entre os isolados de B. bassiana indicando que este fungo representa um agregado de esp?cies. Al?m disso, a dist?ncia gen?tica detectada entre os isolados foi fortemente relacionada a dist?ncia geogr?fica de origem dos isolados. Posteriormente, os isolados tamb?m foram avaliados quanto a toler?ncia ? radia??o UV-B, toler?ncia ao calor e atividade ao frio. Tanto o calor quanto o frio s?o fatores abi?ticos ambientais que restringem o uso de fungos como agentes de biocontrole, enquanto a radia??o solar, particularmente UV-B, ? o maior fator ambiental que pode afetar negativamente a a??o deste entomopat?geno. Neste sentido, foi observada alta variabilidade entre os isolados analisados em rela??o ? toler?ncia a UV-B ap?s duas horas de exposi??o ? radia??o, obtendose percentuais de germina??o de con?dios que variaram entre 0% e aproximadamente 80%. Al?m disso, foi observado atraso na germina??o dos con?dios, e revelado que os isolados originados de baixas latitudes tenderam a apresentar menor toler?nc ia a UV-B do que aqueles provenientes de latitudes mais elevadas. Foi tamb?m encontrada alta variabilidade em termotoler?ncia dos con?dios ap?s duas horas de exposi??o a 45 ?C, variando entre baixa, m?dia e alta viabilidade. Os con?dios mais resistentes n?o toleraram exposi??o entre 44 ?C e 45 ?C por seis horas. Quando submetidos a 5 ?C a maioria dos isolados apresentaram atividade. Os isolados de B. bassiana obtidos de latitudes mais elevadas foram mais ativos ao frio do que aqueles originados pr?ximos ? linha do equador. Al?m disso, os isolados tamb?m foram avaliados quanto ao potencial virulento sobre larvas de Boophilus microplus, e neste sentido foi poss?vel diferenci?-los em baixa, m?dia e alta virul?ncia. Foi detectado tamb?m que diferentes popula??es desta esp?cie de carrapato podem manifestar diferentes n?veis de suscetibilidade ? infec??o por B. bassiana. Neste sentido, o presente estudo permitiu selecionar isolados deste entomopat?geno com maior potencial para o controle biol?gico de B. microplus e outros artr?podes em condi??es de campo.

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.

Zugl.: Hohenheim, Univ., Diss., 2008. / Enthält u.a. vier Abstracts.

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.

Submitted by Nathália Faria da Silva (nathaliafsilva.ufv@gmail.com) on 2017-06-08T13:32:28Z No. of bitstreams: 1 texto completo.pdf: 4048898 bytes, checksum: f5cec7deaee542231b18913fa6a7d0e7 (MD5) / Made available in DSpace on 2017-06-08T13:32:28Z (GMT). No. of bitstreams: 1 texto completo.pdf: 4048898 bytes, checksum: f5cec7deaee542231b18913fa6a7d0e7 (MD5) Previous issue date: 2004-10-08 / Fundação de Amparo a Pesquisa do Estado de Minas Gerais / O carrapato B. microplus é considerado o principal ectoparasita para a pecuária bovina mundial. Seu controle repousa pesadamente sobre a utilização de acaricidas que a cada ano que passa se mostram menos eficientes devido a aquisição de resistência pelo parasita. Como alternativa ecologicamente segura para controlar este parasito, tem sido desenvolvidas vacinas derivadas de antígenos ocultos para o sistema imune do hospedeiro. Porém, falhas vacinais com a glicoproteína Bm86 recombinante já tem sido descritas devido à polimorfismos no seu gene em diferentes populações de B. microplus. Já foi descrito variações de até 8.6% na seqüência deste gene. Uma variante da Bm86, denominada Bm95, foi identificada e também utilizada como vacina recombinante, revertendo os resultados negativos naquelas populações que foram resistentes à vacinação com a Bm86, mas com eficiências variáveis. Pesquisadores do Laboratório de Biologia e Controle de Hematozoários, da Universidade Federal de Viçosa, MG desenvolveram uma vacina sintética composta de três peptídeos derivados da Bm86, sendo que um deles, o 4824, é considerado o principal indutor da produção de anticorpos anti-SBm7462. Com o intuito de avaliar mutações no gene bm86 e a conservação da seqüência peptídica 4824 foram analisadas 30 populações de carrapatos B. microplus, de regiões geograficamente distintas do Brasil e países vizinhos. Para isso foi sintetizado, através do RNA total extraído das amostras, o cDNA que serviu de molde nas reações de PCR para amplificação da seqüência nucleotídica compreendida entre os nucleotídeos 278 – 1071. O material amplificado foi clonado em vetor pGEM ® e seqüenciado em seqüenciador automático pelo método enzimático. A análise foi feita através de alinhamento de múltiplas seqüências pelo programa BioEdit versão 5.0.5 e a verificação de polimorfismos por inspeção visual. As análises confirmaram as conservações das seqüências nucleotídicas e aminoácidicas do peptídeo 4824 e revelaram variabilidade genética dentro do gene bm86 entre as amostras pesquisadas. Através do sequenciamento do fragmento que codifica o peptídeo 4822 foi possível determinar que os testes realizados com a vacina sintética SBm7462 foram em amostras que divergiam em dois aminoácidos da seqüência peptídica da vacina, além de apresentarem variações superiores a 4.94% na seqüência da glicoproteína intestinal. / The tick B. microplus is considered the main ectoparasite for the world bovine cattle. Its control rests heavily about the acaricides use that are less efficient due to resistance acquisition on the part of the parasite to every year that passes. As alternative ecologically holds to control this parasite, it has been developed derived vaccines of cancealed antigens for the immune system of the host. Even so, flaws vaccinate with the protein Bm86 recombinant it has already been described due to the polymorphisms of its gene in different populations of B. microplus. It was already described variations of up to 8.6% in the sequence of this gene. A variant of Bm86, denominated Bm95, it was identified and also used as vaccine recombinant, reverting the negative results in those populations that were resistant to the vaccination with for Bm86 but with variable efficiencies. Researchers of the Laboratory of Biology and Control of Hematozoários, of the Federal University of Viçosa, MG developed a synthetic vaccine composed of three derived peptides of Bm86, and one of them, the 4824, the main inductor of the production of antibodies anti-SBm7462 is considered. With the intuit of evaluating mutations in the gene xiibm86 and the conservation of the sequence peptides 4824, 30 populations of ticks B. microplus were analyzed, of areas geographically different from Brazil and neighboring countries. For that it was synthesized, through extracted total RNA of the samples, the cDNA that served as mold in the reactions of PCR for amplification of the sequence of nucleotides understood among the nucleotides 278 - 1071. The amplified material was cloned in vectorial pGEM® and sequenced in automatic sequencer for the enzymatic method. The analysis was made through alignment of multiple sequences by the program BioEdit version 5.0.5 and the polymorphisms verification for visual inspection. The analyses confirmed the conservations of the sequences nucleotides and amino acids of the peptides 4824 and they revealed genetic variability inside of the gene bm86 among the researched samples. Through the sequencing of the fragment that codes the peptides 4822 it was possible to determine that the tests accomplished with the synthetic vaccine SBm7462 were in samples that diverged in two amino acids of the sequence peptides of the vaccine, besides they present superior variations at 4.94% in the sequence of the intestinal protein.

Submitted by Nathália Faria da Silva (nathaliafsilva.ufv@gmail.com) on 2017-07-19T13:55:20Z No. of bitstreams: 1 texto completo.PDF: 430274 bytes, checksum: a912ed0418d9449f1aa8723bf53fc623 (MD5) / Made available in DSpace on 2017-07-19T13:55:20Z (GMT). No. of bitstreams: 1 texto completo.PDF: 430274 bytes, checksum: a912ed0418d9449f1aa8723bf53fc623 (MD5) Previous issue date: 2002-02-19 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / O peptídeo sintético SBm7462 foi avaliado como imunógeno para o controle do carrapato Boophilus microplus em teste misto (estábulo e campo). O estudo se realizou com as doses de 1,0mg e 2,0mg em 30 bovinos da raça holandesa com média de grau de sangue de 91,16% (H/Z), os quais foram mantidos sob o manejo rotineiro no Campo Experimental do Centro Nacional de Pesquisa em Gado de Leite-EMBRAPA/CNPGL em pastos altamente infestados com larvas dessa espécie de carrapato. Os animais foram separados em três grupos: vacinal 2,0mg, vacinal 1,0mg e controle, sendo os grupos vacinais imunizados nos dias 0, 30 e 60 a campo. Foram realizadas a contagem de carrapatos e a coleta de sangue semanalmente, a partir do dia 0. Duas semanas após a terceira inoculação, os animais foram levados para baias individuais e submetidos a uma infestação artificial com um total de 4.500 larvas/animal de B. microplus (amostra “Porto Alegre”). A partir do 19 o dia pós-infestação, foram coletadas as teleóginas desprendidas, contadas, pesadas, identificadas e incubadas em estufa B.O.D. por quinze dias, quando os respectivos ovos foram pesados. A análise da fertilidade desses ovos e a viabilidade das larvas provenientes foram também analisadas. A cinética da produção de anticorpos foi acompanhada através de ELISA, obtendo- se títulos maiores para os grupos vacinais, estatisticamente diferentes do grupo controle (p<0,01). Os parâmetros biológicos foram analisados levando-se em conta o número de teleóginas, peso dos ovos e fertilidade dos ovos. A eficácia foi de 15,8% para a dose de 1,0mg do peptídeo sintético SBm7462 e de 53,29% para 2,0mg, com diferenças estatísticas significativas entre os grupos 1,0mg, 2,0mg e controle (p<0,05). Quanto à avaliação do número de carrapatos a campo, não houve diferença estatística entre os grupos testados. Os resultados obtidos mostram que o peptídeo sintético SBm7462 é uma alternativa viável para o controle do carrapato comum dos bovinos, pela sua eficácia, segurança e baixo custo. / The synthetic peptide SBm7462 was evaluated as immunogen for the control of the tick Boophilus microplus in mixed test (stable and field). The study was done with the quantity of 1,0mg and 2,0mg of the peptide SBm7462 on 30 Holstein female cattles (91,16% H/Z), which were maintained under the routine handling in the experimental field of the Centro Nacional de Pesquisa em Gado de Leite- EMBRAPA/CNPGL on pastures highly infested with larvaes of that tick species. The animals were separate in three groups: vaccinated with 2,0mg, vaccinated with 1,0mg and control, being the vaccinated groups immunized on the days 0, 30 and 60 in field. The count of the adults females ticks and the blood collection were weekly done, starting from the day 0. Two weeks after the third inoculation, the animals were taken to individual stalls and submitted to an artificial infestation with 4.500 larvaes/animal of B. microplus (strain “Porto Alegre”). Starting on the 19 o . day post-infestation, the adult females ticks were collected, counted, identified, weighed and incubated in B.O.D. After 15 days, the respective eggs were weighed. The analysis of the eggs fertility and the viability of the larvaes were also analyzed. The kinetics of the production of antibodies was accompanied through of ELISA, being obtained larger titles for the vaccinated groups with statistic differences to the control group (p < 0,01). The biological parameters were analyzed through of the account the number of adult females ticks, eggs weight and eggs fertility. The efficacy was of 15,8% for the 1,0mg of the synthetic peptide SBm7462 and of 53,29% for the 2,0mg, of the same peptide, with significant statistical differences among the groups 1,0mg, 2,0mg and control (p < 0,05). When compared the number of ticks in field, there was not statistic difference among the tested groups. The obtained results show that the synthetic peptide SBm7462 is a viable alternative for the control of the cattle tick, regarding its efficacy, safety and low cost.