Made available in DSpace on 2014-08-20T14:37:59Z (GMT). No. of bitstreams: 1 dissertacao_luciana_wendt.pdf: 2823644 bytes, checksum: cf4f4a14caafbe809001eb7426830a04 (MD5) Previous issue date: 2009-07-27 / The parasites of capybaras (Hydrochoerus hydrochaeris) in semi-intensive breeding were searched, and examined 39 capybaras (20 young and 19 adults) for ectoparasites, 34 capybaras (13 young and 21 adults) for gastrointestinal helminths. We estimated the parameters of parasitism: prevalence, abundance and mean intensity. Fifty two fecal samples were examined for identification of Eimeria spp. Ticks collected were Amblyomma dubitatum and immature of Amblyomma sp. Among the helminths, four species were identified in young capybaras, with the prevalence and intensity of infection: Capillaria hydrochoeri (46,15% and 11,5 specimens), Hydrochoerisnema anomalobursata (76,92% and 34,2 specimens), Protozoophaga obesa (46,15% and 6,33 specimens) and Monoecocestus hagmanni (23,07% and 1,66 specimens). In adults were found five species: C. hydrochoeri (66,66% and 16,42 specimens), H. anomalobursata (42,85% and 79,88 specimens), P. obesa (100% and 1655,42 specimens), M. hagmanni (14,28% and 38,33 specimens) and Monoecocestus macrobursatum (4,76% and two specimens). Reports of M. hagmanni and M. macrobursatum parasite capybaras in the state of Rio Grande do Sul are unpublished. P. obesa had significantly higher parameters in adult capybaras. In fecal samples, were identified: Eimeria trinidadensis (88,46%) with oocysts that measured 21,02μm (±1,03μm) length and 19,15 (±1,42μm) wide, and Eimeria ichiloensis (79,92%), with oocysts that measured 25,19μm (±1,77μm) length and 19,7μm (±1,3μm) wide. The diversity of founded parasites show the importance of control programs in semi-intensive breeding of capybaras. / A fauna parasitária de capivaras (Hydrochoerus hydrochaeris) em sistema de criação semi-intensivo no município de Capão do Leão, RS foi pesquisada. Foram examinadas 39 capivaras (20 filhotes e 19 adultas) para ectoparasitos, e 34 capivaras (13 filhotes e 21 adultas) para helmintos gastrintestinais, sendo estimados os parâmetros de parasitismo: prevalência, abundância e intensidade média. Também 52 amostras fecais foram analisadas para identificação de Eimeria spp. Observou-se adultos de Amblyomma dubitatum e imaturos de Amblyomma sp. Quatro espécies de helmintos foram identificadas nas capivaras filhotes, com a seguinte prevalência e intensidade de infecção: Capillaria hydrochoeri (46,15% e 11,5 espécimes), Hydrochoerisnema anomalobursata (76,92% e 34,2 espécimes), Protozoophaga obesa (46,15% e 6,33 espécimes), e Monoecocestus hagmanni (23,07% e 1,66 espécimes). Nas capivaras adultas houve cinco espécies: C. hydrochoeri (66,66% e 16,42 espécimes), H. anomalobursata (42,85% e 79,88 espécimes), P. obesa (100% e 1655,42 espécimes), M. hagmanni (14,28% e 38,33 espécimes) e Monoecocestus macrobursatum (4,76% e dois espécimes). Os relatos de M. hagmanni e de M. macrobursatum, parasitando capivaras no estado do Rio Grande do Sul são inéditos. P. obesa apresentou parâmetros significativamente superiores nas capivaras adultas. Nas amostras fecais identificou-se: Eimeria trinidadensis (88,46%) com oocistos subesféricos que mediram em média 21,02μm (±1,03μm) de comprimento e 19,15μm (±1,42μm) de largura, e Eimeria ichiloensis (79,92%), com oocistos que mediram em média 25,19μm (±1,77μm) de comprimento e 19,7μm (±1,3μm) de largura. A diversidade de parasitos encontrados evidencia a necessidade de medidas de controle em criações semi-intensivas de capivaras.

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

The recent discovery of Amblyomma maculatum sensu lato (s. l.) ticks in southern Arizona has renewed discussions around species designations for members of the Amblyomma maculatum tick group. Amblyomma maculatum s. l. from Arizona appear to be morphologically intermediate between A. maculatum sensu stricto (s. s.) and A. triste s. s. At present there is no conclusive species designation for the ticks from Arizona. My research focused on analyzing the systematics of both A. maculatum s. l. and Rickettsia parkeri, a common bacterial pathogen transmitted by these ticks. In the laboratory, A. maculatum s. l. from Arizona and A. maculatum s. s. from Georgia readily mated on experimental animals to produce F1 hybrid ticks; there was no difference in fertility with these two populations when compared with homologous populations. However, the F1 hybrids produced during these experiments exhibited diminished fitness and did not produce a viable F2 generation. These results suggest that A. maculatum s. l. and A. maculatum s. s. represent separate biological species. Results of the crossbreeding experiment conflict with recent genetic analyses of A. maculatum s. l. and A. maculatum s. s. suggesting they are a single species. Thus, I developed and optimized 14 microsatellite loci that amplify both A. maculatum s. s. and A. maculatum s. l. These novel microsatellite markers can be used in future analyses of A. maculatum s. l. and A. maculatum s. s. to further test for conspecificity between the two. I also investigated the genetic relationships within geographically distinct R. parkeri strains through development and implementation of a multi-locus sequence typing analysis. I showed that while there is no consistent genetic delineation of strains isolated from A. maculatum s. l. versus A. maculatum s. s., there is a subset of R. parkeri strains from A. maculatum s. l. that appear to represent an intermediate genotype between the North and South American strains. While the biological causes for these results are not immediately clear, coevolution of R. parkeri and A. maculatum s. l. may account for the detection of the intermediate genotype only found in association with A. maculatum s. l.

Made available in DSpace on 2014-07-29T15:29:20Z (GMT). No. of bitstreams: 1 Edmeia de paula.pdf: 2653466 bytes, checksum: ad9e1a87d15b9af7be8e8cea4757bf4f (MD5) Previous issue date: 2010-04-15 / Brazilian Spotted Fever (BSF) is an acute, infectious, febrile disease of varying severity, its course ranging from asymptomatic cases to severe clinical forms with a high lethality rate. The absence of pathognomonic signs in this anthropozoonosis often results in late diagnosis, thereby serving as a stimulus for investigators to focus their attention on the weakest link of its epidemiological chain, the vector. In Brazil, the principal vector of Rickettsia rickettsii, the etiological agent of spotted fever in the New World, is the ixodid tick Amblyomma cajennense. In the present study, a systematic literature review was performed to search for publications on the epidemiology of BSF in the country. With the objective of improving control of the vector, its resistance to the principal acaricides applied against this tick was monitored. The systematic literature review was performed by searching for scientific papers dealing with BSF in the Cochrane Library, LILACS, MEDLINE and SciELO databases, using the keywords Amblyomma cajennense, Brazilian spotted fever and Rickettsia rickettsii. The search included articles published in English and Brazilian Portuguese that contained these keywords. To evaluate susceptibility and resistance to the synthetic acaricides, larvae had to be obtained from engorged female specimens of A. cajennense collected from naturally infested horses that had been free of acaricidal residue for at least 45 days, on farms belonging to five municipalities of the state of Goiás (Caldas Novas, Hidrolândia, Goiás, Terezópolis and Goiânia). The larvae were exposed to different concentrations of 12 commercially available acaricidal formulations using the larval packet test (LPT). The possible development of resistance of adult specimens of A. cajennense to the pyrethroid deltamethrin, the principal acaricide on the market in Goiás for the control of ticks in the region, was also evaluated. Unfed adult male and female specimens of A. Cajennense were collected on leaves of bushes along a nature trail in the municipality of Caldas Novas, Goiás, Brazil. They were immersed in different concentrations of deltamethrin and then placed in contact with filter paper impregnated with the substance. The control groups were exposed to distilled water alone. The bioassays were performed in quadruplicate at a temperature of 27oC, relative air humidity >80% and 12-hour light/dark cycles. From the systematic literature review, it was found that there are no records or even systematic studies of BSF in Brazil up to the present moment and the number of cases reported in the literature is lower than the figures published by the health surveillance authorities of the Ministry of Health. The data collected in this review should serve to stimulate investigators to design new studies and carry out further work, since the distribution of this disease may be more widespread than is currently supposed. Results from the bioassays showed a mean percentage of mortality ( ) of 72.6% of the adult specimens after 24 hours of exposure to the dose of deltamethrin prescribed by the manufacturer, characterizing a status of resistance to deltamethrin. The males were found to be more susceptible to this pyrethroid compared to the females. of 82%, 89%, 89.6% and 90% of the larvae were obtained, respectively, with deltamethrin, cypermethrin + piperonyl butoxide, amitraz and permethrin, characterizing a status of probable resistance of the larvae to these acaricides. The combination of cypermethrin with citronellal and with piperonyl butoxide did not significantly affect the lethal capacity of this pyrethroid against the larvae of this vector. Significant mortality was not found in the control groups (p = 0.05). These results encourage new studies to be conducted to develop alternative methods of integrated control of this vector that would be more effective and have less harm on the environment. / A Febre Maculosa Brasileira (FMB) é uma doença infecciosa febril aguda, de gravidade variável, podendo cursar desde formas assintomáticas até formas graves, com elevada taxa de letalidade. A ausência de sinais patognomônicos nesta antropozoonose, que muitas vezes leva ao seu diagnóstico tardio, suscita o ataque do elo mais fraco de sua cadeia epidemiológica seu vetor. No Brasil, o principal vetor da Rickettsia rickettsii, agente etiológico da Febre Maculosa da Região Neotropical é o ixodídeo Amblyomma cajennense. Neste trabalho foi feita uma revisão sistemática da literatura abordando a temática da epidemiologia da FMB no Brasil. Com finalidade de subsidiar o controle do vetor monitorou-se a resistência do mesmo aos principais produtos acaricidas utilizados em seu controle. A revisão sistemática da literatura foi feita utilizando artigos científicos que abordam a temática da FMB no Brasil, pesquisando nas bases de dados Biblioteca Cochrane, LILACS, MEDLINE e SciELO. Utilizando como descritores Amblyomma cajennense; Febre Maculosa; Rickettsia rickettsii. Participaram da revisão artigos publicados na língua inglesa e portuguesa do Brasil que apresentaram os descritores. Na parte experimental para avaliar a suscetibilidade e resistência dos acaricidas químicos sintéticos, fo ram obtidas larvas de teleóginas de A. cajennense. Estas foram coletadas de cavalos naturalmente infestados e livres de resíduos acaricidas há pelo menos 45 dias, em fazendas pertencentes a cinco municípios do Estado (Caldas Novas, Hidrolândia, Goiás, Terezópolis e Goiânia). As larvas foram expostas a diferentes concentrações das 12 formulações acaricidas comerciais, pelo método de Larval Packet Test (LPT). Para verificar o possível desenvolvimento de resistência de adultos de A. cajennense ao piretroide deltametrina, principal base carrapaticida comercializada em Goiás para o controle de carrapatos na região. Machos e fêmeas, adultos, de A. cajennense em jejum foram coletados em folhas de arbustos em uma trilha ecológica, no município de Caldas Novas, Goiás, Brasil. Estes foram submetidos a diferentes concentrações de deltametrina, através de imersão e posterior contato com papel filtro impregnado. Os grupos controles foram submetidos apenas à água destilada. Os bioensaios foram realizados em quadruplicata, a 27ºC, UR > 80% e 12h de luz. Através da Revisão Sistemática da Literatura verificou-se que não há registros ou mesmo estudos sistemáticos da Febre Maculosa no Brasil; até o momento o número de casos relatados na literatura são inferiores aos que a Secretaria de Vigilância em Saúde do Ministério da Saúde tem divulgado. Os dados levantados pela revisão sugerem que novas investigações e estudos sejam feitos, pois a distribuição desta doença pode ser mais ampla do que se supõe. Foi observado, nos bioensaios, que a mortalidade média percentual ( ) de 72,6% dos adultos após 24 h de exposição à dosagem de deltametrina prescrita pelo fabricante, caracterizando o status de resistência à deltametrina. Os machos mostraram-se mais suscetíveis que as fêmeas ao piretroide. de 82%, 89%, 89.6% e 90% das larvas foram obtidas, respectivamente, por deltametrina; cipermetrina + butóxido de piperolina; amitraz e permetrina, caracterizando o status de provável resistência larval a estes acaricidas. As associações de cipermetrina com citronelal e com butóxico de piperolina não interferiram significativamente na capacidade letal desse piretroide para larvas do vetor. Mortalidade significativa não foi evidenciada nos grupos controles (p = 0,05). Os resultados suscitam a realização de estudos para o desenvolvimento de medidas alternativas de controle integradas, mais eficientes e de menor impacto ambiental, para o controle deste vetor.

With advances in molecular technology, the study of human ageing has turned to DNA for answers as to how humans age. Due to the size of the human genome and the longevity of humans, organisms with smaller genomes and shorter lifespans have frequently been the center of research studies in ageing. Studies of Drosophila melanogaster, Caenorhabditis elegans, yeast, and mice have uncovered specific genes that up and down regulate with age and stress. Research has yet to produce, however, results from an organism known for its longevity. Amblyomma americanum is an excellent candidate for this, as it can survive for years unfed. Two groups of 75 unfed adult A. americanum were monitored in a control environment of 85% relative humidity and an experimental environment designed to induce physiological stress at 75% relative humidity. Five ticks were tested for transcript abundance of five candidate ageing genes initially and at the 25th, 75th, and 95th percent mortality. These tests provided evidence that Amblyomma americanum undergoes changes in gene expression with age on a genetic level.

With advances in molecular technology, the study of human ageing has turned to DNA for answers as to how humans age. Due to the size of the human genome and the longevity of humans, organisms with smaller genomes and shorter lifespans have frequently been the center of research studies in ageing. Studies of Drosophila melanogaster, Caenorhabditis elegans, yeast, and mice have uncovered specific genes that up and down regulate with age and stress. Research has yet to produce, however, results from an organism known for its longevity. Amblyomma americanum is an excellent candidate for this, as it can survive for years unfed. Two groups of 75 unfed adult A. americanum were monitored in a control environment of 85% relative humidity and an experimental environment designed to induce physiological stress at 75% relative humidity. Five ticks were tested for transcript abundance of five candidate ageing genes initially and at the 25th, 75th, and 95th percent mortality. These tests provided evidence that Amblyomma americanum undergoes changes in gene expression with age on a genetic level.

The interaction of tick species and the microclimate that they inhabit is a subject that is frequently studied. The known biology of the Gulf Coast tick, Amblyomma maculatum Koch (Acari: Ixodidae), was used to conduct analyses to determine which meteorological factors regulate Gulf Coast tick survivorship in an area of Texas that is known for reduced occurrence of this tick species. Gulf Coast tick collection records, that indicated the collection of a single tick or multiple ticks from one animal or multiple animals at livestock markets, were obtained from the Texas Animal Health Commission. These records were used as an indicator of adult Gulf Coast tick abundance during each year in the 90 county study area and were used as the dependent variable in linear, quadratic, and cubic regression analyses. Independent variables used in these analyses were precipitation and differing drought thresholds during the peak activity time of the four life stages of the Gulf Coast tick and during combined life stage peak activity times. Linear, quadratic, and cubic regression analyses to measure the effect of precipitation during differing peak activity times of the Gulf Coast tick on adult Gulf Coast tick collection records were not statistically significant. These three regression analyses were also used to measure the effect of increasing drought thresholds, measured using a Keetch-Byram Drought Index, on adult Gulf Coast tick collection records. A determination was made that increasing drought thresholds during the peak activity time of differing Gulf Coast tick life stages reduce the number of Gulf Coast tick collection records the following year.

Feral swine (Sus scrofa domesticus) are spreading across North America at an alarming rate. Four Canadian provinces and 39 states within the continental United States now report standing populations of feral pigs. Estimates place the number of feral hogs in Texas >2M, accounting for more than half of the United States population. It is known that feral swine impact local ecology following establishment, with regard to shifts in local flora and fauna. The overall objective of this research was to investigate the role of feral swine in tick-borne pathogen transmission in Texas. The underpinning objectives were to establish host records for tick species parasitizing feral swine, determine the species assemblages associated with feral swine among different ecoregions of Texas, determine by immunoassay to which tick-borne bacteria feral pigs were being exposed, and detect the DNA of tick-borne bacteria by polymerase chain reaction assay in the event of poor or early immune response by the host. Feral pigs (N=432) were harvested from June 2008 to June 2010 using box and corral traps and by aerial gunning. Seven species of ticks, Amblyomma americanum, A. cajennense, A. maculatum, Dermacentor albipictus, D. halli, and D. variabilis; and Ixodes scapularis, were collected. Immature stages of A. cajennense and A. americanum were collected as well. All classes of feral pigs, gender by age, were infested with ticks. Serum was collected through a multi-organizational effort from 2006 to 2010 and tested by ELISA for previous exposure to tick-borne pathogens in the genera Rickettsia and Ehrlichia (N=888) and Borrelia (N=849). Prevalence percentages by immunoassay were 27.59 percent, 13.18 percent and 2.12 percent for Rickettsia, Ehrlichia, and Borrelia, respectively. Samples positive by ELISA for exposure to Borrelia were further screened by Western Blot for exposure to Borrelia turicatae. The results were equivocal in most cases. Blood samples (N=233) were collected from 2008 to 2010 and analyzed by polymerase chain reaction for the detection of the DNA of these same three genera of bacteria. Two of the samples were positive by PCR for the presence of Borrelia DNA. Texas feral swine are serving as hosts for at least seven species of ticks and are interacting with tick-borne pathogen transmissions cycles in Texas.

The aggregation-attachment-pheromone (AAP) of two geographic strains of the Gulf Coast tick, Amblyomma maculatum Koch, was investigated to evaluate practicality of using solid-phase-microextraction (SPME) in an AAP study of Gulf Coast tick. Solid-phase microextraction was used to compare the AAP production in two strains of fed male Gulf Coast tick and demonstrate and confirm the presence of AAP in bioassays. A solid-phase-microextraction (SPME) headspace collection technique was sufficient to capture volatile organic compounds produced by fed and unfed male Gulf Coast ticks. Gas chromatography analysis revealed three major volatile organic compounds were produced in significantly greater amounts (p < 0.05) by fed males than those produced by unfed males. These volatile compounds were produced in significantly higher amount by the third day of feeding by male ticks. However, two of these volatiles remained relatively constant in their production while the primary volatile compound increased in its production until the eighth day of feeding by male Gulf Coast ticks. Also, the relative abundances of these three volatile organic compounds were different between Oklahoma and Texas strains of Gulf Coast ticks. The activity of AAP from fed male Gulf Coast ticks was confirmed using two bioassay techniques. A petri dish bioassay revealed significantly higher numbers of female Gulf Coast ticks attracted to fed-males which also produced significantly greater amounts (p < 0.001) of volatile organic compounds detected by GC analysis. The Ytube olfactometer bioassay revealed that significantly higher numbers of females responded to fed-males or to CO2 when compared to purified air (p < 0.001), but the differences in female response to fed-males and CO2 were not significant (p < 0.391 in Oklahoma strain and p < 0.458 in Texas strain). However, female responses to stimuli containing both fed-males and CO2 were significantly higher when compared to either stimulus alone (p < 0.001).

Infestation of cattle by Lone Star ticks, Amblyomma americanum, leads to damage of hides intended for leather, weight loss, infertility, and potentially death of cattle, which contribute to production losses for farmers. Public concerns regarding chemical residues in food and the environment necessitate development of chemical-free alternative tick controls, such as breeding for tick-resistant phenotypes and developing anti-tick vaccines. Thus, the goal of this study was elucidation of mechanisms that mediate immune responses in cattle infested with A. americanum using gene expression techniques. Methods for isolation of total RNA from bovine tick bite-site biopsies and blood leukocytes were optimized to provide RNA suitable for gene expression studies. Tick bite-site biopsies (6 mm) and blood leukocytes were collected from a total of 13 calves (N=6, Group 4 and N=7, Group 5 calves) during experimental tick infestations to determine A. americanum tick-susceptible and -resistant phenotypes. Microarray experiments compared gene expression in tick bite-sites from tick-susceptible, moderately tick-resistant, and highly tick-resistant calves. A total of 35 genes were profiled in tick bite-site biopsies and 12 genes were evaluated in blood leukocytes via gene-specific qRT-PCR assays, and analyzed for each phenotype and for each group of calves as a whole. Analysis of microarray data revealed differential expression of IL-1R-mediators among the three cattle phenotypes. Expression profiles generated by qRT-PCR for TLR-mediating genes such as TLR2, TLR4, CD14, and MyD88 suggest that a MyD88-dependent signaling pathway may mediate the development of acquired immunity in cattle infested with Lone Star ticks. Additionally, increased expression for IL12, IFNgamma, and TNFalpha suggests that a Th1-type cell-mediated reaction may be activated, whereas increased expression of IL6, IL10, and IGHG1 supports the involvement of a Th2-type humoral-mediated response at tick bite-sites in cattle infested with at A. americanum. Regression analyses identified strong correlations between factors involved in pattern recognition in tick bite-site biopsies, including associations between TLR4 and IL1alpha, and between IL1alpha and IL1RN. In conclusion, this dissertation reports optimal methodology for gene expression studies in tick-infested cattle and provides preliminary data concerning the underlying mechanisms associated with the immune response in Lone Star tick-infested cattle.