Orientador: Raimundo Souza Lopes / Coorientador: Pedro Paulo Pires / Banca: Regina Kiomi Takahira / Banca: Cecília Braga Laposy / Resumo: O estudo teve como objetivo verificar possíveis alterações hematológicas e bioquímicas que ocorrem no sangue bovino de animais hígidos e parasitados com Anaplasma marginale, conservado por 21 dias em bolsas plásticas contendo Citrato-Fosfato-Dextrose-Adenina (CPDA-1) e avaliar a ação da Riboflavina associada à radiação ultravioleta (UV) na inativação do hemoparasita. Na primeira etapa foram determinados o número de Hemácias (He) e Leucócitos, Hemoglobina (Hb), Volume Globular (VG), Volume Corpuscular Médio (VCM), Proteína Plasmática Total (PPT), Fibrinogênio, Sódio (Na+), Potássio (K+) e Lactato, a cada três dias, durante 21 dias, do grupo I formado por bolsas contendo sangue de bovinos saudáveis e do grupo II, por bolsas com sangue parasitado por A. marginale. Na segunda, foram realizados os exames do Esfregaço Sanguíneo Corado (ESC), Reação de Imunofluorescência Indireta (RIFI) e Reação em Cadeia da Polimerase quantitativa (qPCR) antes e após o tratamento com Riboflavina associada à radiação UV, além da inoculação em bezerros para verificar se o parasita mantinha sua infectividade. Estes exames foram repetidos in vivo nos dias sete, 14 e 21 após a inoculação. Na primeira etapa, para ambos os grupos, ocorreu redução dos valores de He, Hb, VG, PPT, Leucócitos e Na+; e aumento no VCM, K+ e Lactato. Na segunda etapa, ocorreu redução da carga parasitária de 30,60% no momento zero (M0) para 15,26% após o tratamento; RIFI de 100% positivo no M0, para 100% negativo após o tratamento. A qPCR demonstrou que o tratamento não promoveu completa eliminação do parasita, mas houve uma inativação parcial, já que os bezerros receptores não adoeceram / Abstract: The objective of this study was to evaluate hematological and biochemical changes in bovine blood from healthy and Anaplasma marginale infected animals after a 21day storage period in plastic bags with Citrate- Phosphate-Dextrose-Adenine (CPDA-1) and also to evaluate the effect of Riboflavin associated with ultraviolet radiation (UV) on the inactivation of the hemoparasite. In the first experimental phase, the number of Erythrocytes (RBC) and Leukocytes, Hemoglobin (Hb), Globular Volume (GV), Mean Corpuscular Volume (MCV), Total Plasma Protein (TPP), Fibrinogen, Sodium (Na+), Potassium (K+) and Lactate was determined every 3 days for 21 days in blood stored in plastic bags from clinically healthy animals (Group I) and in blood stored in plastic bags containing blood from infected animals by Anaplasma marginale (Group II). In the second experimental phase, stained blood smears, indirect immunofluorescence assay (IFA) and quantitative polymerase chain reaction (qPCR) were performed before and after the treatment with riboflavin associated with UV radiation. Also, calves were inoculated to evaluate the infectivity of the parasite. These exams were repeated at day 7, 14 and 21 after inoculation. In the first phase a reduction in RBC, Hb, GV, TPP, Leukocytes and Na+ and an increase in MCV, K+ and Lactate were observed in both groups. In the second phase, the parasite load of 30.60% at time zero (T0) was reduced to 15.26% after the treatment; IFA of 100% positive at T0 was reduced to 100% negative after the treatment. qPCR revealed that total elimination of the parasite by the treatment was not observed but there was a partial inactivation was observed since the recipient calves did not get sick / Mestre

Made available in DSpace on 2014-06-11T19:30:11Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-03-28Bitstream added on 2014-06-13T18:06:40Z : No. of bitstreams: 1 lopes_lg_me_botfmvz.pdf: 686515 bytes, checksum: 3ad48498b96d5ac5f9774f231d0a6ff9 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O estudo teve como objetivo verificar possíveis alterações hematológicas e bioquímicas que ocorrem no sangue bovino de animais hígidos e parasitados com Anaplasma marginale, conservado por 21 dias em bolsas plásticas contendo Citrato-Fosfato-Dextrose-Adenina (CPDA-1) e avaliar a ação da Riboflavina associada à radiação ultravioleta (UV) na inativação do hemoparasita. Na primeira etapa foram determinados o número de Hemácias (He) e Leucócitos, Hemoglobina (Hb), Volume Globular (VG), Volume Corpuscular Médio (VCM), Proteína Plasmática Total (PPT), Fibrinogênio, Sódio (Na+), Potássio (K+) e Lactato, a cada três dias, durante 21 dias, do grupo I formado por bolsas contendo sangue de bovinos saudáveis e do grupo II, por bolsas com sangue parasitado por A. marginale. Na segunda, foram realizados os exames do Esfregaço Sanguíneo Corado (ESC), Reação de Imunofluorescência Indireta (RIFI) e Reação em Cadeia da Polimerase quantitativa (qPCR) antes e após o tratamento com Riboflavina associada à radiação UV, além da inoculação em bezerros para verificar se o parasita mantinha sua infectividade. Estes exames foram repetidos in vivo nos dias sete, 14 e 21 após a inoculação. Na primeira etapa, para ambos os grupos, ocorreu redução dos valores de He, Hb, VG, PPT, Leucócitos e Na+; e aumento no VCM, K+ e Lactato. Na segunda etapa, ocorreu redução da carga parasitária de 30,60% no momento zero (M0) para 15,26% após o tratamento; RIFI de 100% positivo no M0, para 100% negativo após o tratamento. A qPCR demonstrou que o tratamento não promoveu completa eliminação do parasita, mas houve uma inativação parcial, já que os bezerros receptores não adoeceram / The objective of this study was to evaluate hematological and biochemical changes in bovine blood from healthy and Anaplasma marginale infected animals after a 21day storage period in plastic bags with Citrate- Phosphate-Dextrose-Adenine (CPDA-1) and also to evaluate the effect of Riboflavin associated with ultraviolet radiation (UV) on the inactivation of the hemoparasite. In the first experimental phase, the number of Erythrocytes (RBC) and Leukocytes, Hemoglobin (Hb), Globular Volume (GV), Mean Corpuscular Volume (MCV), Total Plasma Protein (TPP), Fibrinogen, Sodium (Na+), Potassium (K+) and Lactate was determined every 3 days for 21 days in blood stored in plastic bags from clinically healthy animals (Group I) and in blood stored in plastic bags containing blood from infected animals by Anaplasma marginale (Group II). In the second experimental phase, stained blood smears, indirect immunofluorescence assay (IFA) and quantitative polymerase chain reaction (qPCR) were performed before and after the treatment with riboflavin associated with UV radiation. Also, calves were inoculated to evaluate the infectivity of the parasite. These exams were repeated at day 7, 14 and 21 after inoculation. In the first phase a reduction in RBC, Hb, GV, TPP, Leukocytes and Na+ and an increase in MCV, K+ and Lactate were observed in both groups. In the second phase, the parasite load of 30.60% at time zero (T0) was reduced to 15.26% after the treatment; IFA of 100% positive at T0 was reduced to 100% negative after the treatment. qPCR revealed that total elimination of the parasite by the treatment was not observed but there was a partial inactivation was observed since the recipient calves did not get sick

Submitted by Reginaldo Soares de Freitas (reginaldo.freitas@ufv.br) on 2018-06-28T17:20:50Z No. of bitstreams: 1 texto completo.pdf: 1170314 bytes, checksum: aef38342ffa69b1c377766d841c96e8c (MD5) / Made available in DSpace on 2018-06-28T17:20:50Z (GMT). No. of bitstreams: 1 texto completo.pdf: 1170314 bytes, checksum: aef38342ffa69b1c377766d841c96e8c (MD5) Previous issue date: 2018-02-23 / Neste estudo foram analisadas 100 amostras de sangue de cães, com trombocitopenia marcada (plaquetas < 100.000/μL) ou com visualização direta de hemoparasitas (ou inclusões compatíveis) no esfregaço sanguíneo, obtidas da rotina do laboratório de análises clínicas do DVT (Departamento de Veterinária), do Hospital Veterinário da Universidade Federal de Viçosa (UFV). As amostras foram submetidas à extração de DNA e posteriormente à reação em cadeia da polimerase de tipo nested (nPCR), para o diagnóstico molecular de A. phagocytophilum e outros hemoparasitas. Do total das amostras, 60 foram positivas para no mínimo um hemoparasita. Foi possível detectar a presença de quatro gêneros de hemoparasitas nas amostras de sangue avaliadas: Ehrlichia, Anaplasma, Babesia e Hepatozoon. Foram amplificadas um total de 26 produtos do primer Ehrlichia spp monocítica, 23 de Ehrlichia spp granulocítica 26 com BAB-rum e 10 utilizando o hsp70. Logo ao analisar os resultados, de um total de 86 amostras foi possível identificar os seguintes hemoparasitas: 23 E. canis (identidade 94%-100%), 7 A. platys (identidade 97%-100%), 11 B. vogeli (identidade 86%-99%) e 15 H. canis (identidade 94%-99%). Nenhuma amostra foi positiva utilizando o primer msp4, específico de A. phagocytophilum, porém, o sequenciamento de uma das amostras amplificadas teve uma identidade de 99% com um fragmento da sequência parcial do gene 16S rRNA de A. phagocytophilum depositada no GenBank. A não amplificação de produtos com um primer específico para A. phagocytophilum pode ser atribuída a diversas hipóteses, mas isto não exclui a possibilidade que a bactéria esteja presente no município de Viçosa ou da microrregião. / In this study were analyzed blood samples from 100 dogs, with marked thrombocytopenia (platelets/μL < 100,000) or with direct visualization of hemoparasites (or compatible inclusions) in the blood smear, obtained from the routine of the clinical analysis laboratory, of the DVT (Veterinary Department), from the Veterinary Hospital da Universidade Federal de Viçosa (UFV). The samples were submitted for DNA extraction and subsequently to polymerase chain reaction of nested type (nPCR), for the molecular diagnosis of A. phagocytophilum and other hemoparasites. From all the samples, 60 were positive for at least one hemoparasite. It was possible to detect the presence of four genera of hemoparasites in the blood samples assessed: Ehrlichia, Anaplasma, Babesia and Hepatozoon. Were amplified a total of 26 products from the monocytic ehrlichia spp primer, 23 from the granulocytic ehrlichia spp primer, 26 with BAB-rum primer and 10 using the hsp70 primer. After analyzing the results, from a total of 85 it was possible to identify the following hemoparasites: 23 E. canis (identity 94%- 100%), 7 A. platys (identity 97%-100%), 11 B. vogeli (identity 86%-99%) and 15 H. canis (94% -99% identity). No samples were positive using the msp4 primer, specific for A. phagocytophilum, however, the sequence of one of the amplified samples had a 99% identity with a partial sequence of the 16S rRNA gene of A. phagocytophilum deposited on GenBank. The non- amplification of products with a specific primer for A. phagocytophilum can be attributed to several hypotheses, but this does not exclude the possibility that the bacterium is present in the municipality of Viçosa or in the microregion.

Made available in DSpace on 2014-08-20T14:38:04Z (GMT). No. of bitstreams: 1 dissertacao_alvaro_carlos_menezes_arteche.pdf: 1268541 bytes, checksum: 48cb8939395a534c09b30fdb0ec17ef9 (MD5) Previous issue date: 2011-11-21 / This study analyzed, by reaction to indirect immunofluorescence and individual weighting, the levels of protection and weight-gain in beef calves from birth to the age of eight month, which received two doses of vaccine against tick fever and were maintained free from ticks, compared to naturally infested calves in extensive management conditions with continuous grazing in a rural property situated in the municipality of Santana do Livramento Espinilho / RS. Two groups were randomly gathered, n=30, from which Group I (test) was kept free from ticks from birth to weaning and received two doses of the attenuated, trivalent, refrigerated vaccine against tick fever. Groupe II (control) followed the property s traditional management, which reflects the one used in the region, allowing the tick infestation by the animals. Weight control was accomplished in the first day (d0) and in the last day of the experiment (d180). The serology revealed that 100% of the vaccinated animals presented titles equal to 1:5120 for the three parasites Anaplasma marginale, Babesia bovis e Babesia bigemina (p≤0,025), whereas in the control group the highest titles were 1:2560 in four animals for A. marginale, 1:1280 in four animals for B. bovis and 1:1280 in two animals for B. bigemina (p≤ 0,025). Group I got a mean weight-gain of 30,5 kg more than the control group at the end of the experiment (p≤0,025). The results obtained show that keeping animals free from tick, from birth to weaning, and maintaining them vaccinated against tick fever is safer, more efficient and economically and technically more advantageous than the traditional management system carrapateamento (natural tick infestation). / Avaliaram-se, pela reação de imunofluorescência indireta (RIFI) e de pesagens individuais, os níveis de proteção e ganho de peso de terneiros de corte do nascimento ao desmame com oito meses de idade que receberam duas doses de vacina contra tristeza bovina e foram mantidos livres de carrapatos em comparação a terneiros naturalmente infestados em condições de manejo extensivo com pastoreio contínuo em propriedade rural localizada no município de Santana do Livramento-Espinilho/RS. Formaram-se dois grupos aleatóriamente, n=30, sendo o GRUPO I (teste) mantido livre de carrapatos desde o nascimento até o desmame e com duas doses da vacina atenuada, trivalente e refrigerada contra tristeza bovina. O GRUPO II, controle, seguiu o manejo tradicional da propriedade, que reflete o da região, permitindo que os animais fossem infestados por carrapatos. As pesagens foram realizadas no primeiro dia do experimento (d0) e no último dia do experimento (d180). A sorologia revelou que 100% dos animais vacinados apresentaram títulos maiores ou iguais a 1:5120 para os três parasitos Anaplasma marginale, Babesia bovis e Babesia bigemina (p≤0,025) enquanto que no grupo controle os títulos máximos foram 1:2560 em quatro animais para A. marginale, 1:1280 em quatro animais para B. bovis e 1:1280 em dois animais para B. bigemina. Houve diferença significativa entre os dois grupos (p≤0,025). O grupo I obteve 30,5 Kg a mais de ganho médio de peso que o grupo controle no final do experimento (p≤0,025). Com os resultados obtidos, conclui-se que é mais eficiente, seguro e vantajoso econômica e tecnicamente manter os animais livres de carrapatos, desde o nascimento até o desmame, e vacinados contra tristeza bovina, do que com o manejo tradicional carrapateamento .

Borrelia burgdorferi sensu lato and Anaplasma phagocytophilum are causative agents of Lyme disease and human granulocytic anaplasmosis. Their common vector in Europe are the ticks from the genus Ixodes. In our work, we focused on interaction of innate immune cells with the causative agent of Lyme diseases, that are insubstitutable in their function in the early phase of the disease. Anaplasma phagocytophilum is hard to cultivate, the only possibility is to cultivate it in cell cultures. Successful cultivation of Anaplasma phagocytophilum acquired from patients in our geographic area is crucial for following experiments and for diagnostics too. In our experiments, we used validated cell cultures of HL-60 cells, canine monocytes DH82 and murine monocytes P388D1. During our studies of interaction of the causative agent of Lyme diseases with cells, we used two strains of different species Borrelia. Borrelia garinii M192 and Borrelia burgdorferi sensu stricto B31. These strains vary in virulence. The strain M192 is virulent, but the strain B31 lost its virulence by passages. We specialised in study of morphological changes using light microscopy (observation of dyed and fixed preparates and observation in dark field), eventually by transmision electron microscopy. During our experiments, we concluded that HL-60...

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No description available.

Anaplasma phagocytophilum and A. marginale are the etiologic agents of bovine anaplasmosis and human granulocytic anaplasmosis, respectively. As obligate intracellular pathogens, binding and entry of host cells is a prerequisite for survival. The molecular events associated with these processes are poorly understood. Identifying the adhesins mediating binding, delineating their key functional domains, and determining the molecular determinants to which they bind not only benefits better understanding of Anaplasma spp. pathobiology, but could also benefit the development of novel approaches for protecting against infection. We previously demonstrated that A. phagocytophilum outer membrane protein A (ApOmpA) is critical for bacterial binding and entry host through recognition of α2,3-sialic acid and α1,3-fucose of its receptors, including 6-sulfo-sLex. In this study, we determined that two amino acids, G61 and K64, within its binding domain (ApOmpA59-74), are essential for ApOmpA function. We also confirmed the ability of ApOmpA to act as an adhesin and invasin as it conferred adhesiveness and invasiveness to inert beads. We next extended our studies to A. marginale as it also expresses OmpA (AmOmpA) and its role in infection has not been studied. Molecular models of ApOmpA and AmOmpA were nearly identical, especially in the ApOmpA binding domain and its counterpart in AmOmpA. Antisera raised against AmOmpA or its putative binding domain inhibit A. marginale infection. AmOmpA G55 and K58 are contributory and K59 is essential for AmOmpA to bind to host cells. AmOmpA binding is dependent on α2,3-sialic acid and α1,3-fucose. Coating inert beads with AmOmpA conferred the ability to bind to and be taken up by host cells, confirming that it acts as an adhesin and invasin. 6-sulfo-sLex is dispensable for AmOmpA binding and A. marginale infection. ApOmpA works cooperatively with Asp14 (14-kDa A. phagocytophilum surface protein) to promote optimal infection of host cells. We found that Asp14 is conserved across A. phagocytophilum strains and in A. marginale and confirmed the ability of Asp14 to act as an adhesin and invasin as it conferred adhesiveness and invasiveness to inert beads. Collectively, this work advances our understanding of A. phagocytophilum and A. marginale adhesion and invasion of host cells.

Globally, vector borne diseases cause more than a million deaths each year and more than a billion infections in humans. Ticks are of big medicinal importance since they can transmit pathogens that can cause serious infections. Some recently discovered pathogens that can cause infections in humans are Anaplasma phagocytophilum (A. phagocytophilum) that can cause human granulocytic anaplasmosis (HGA) and Candidatus Neoehrlichia mikurensis (N. mikurensis) that can cause Neoehrlichiosis. It is still widely unknown how prevalent these pathogens are, if ticks can be infected with both of these pathogens and if these pathogens can be transovarially transmitted from adult female to egg and larvae. This study aims to screen for these pathogens in collected ticks from southern Sweden and to detect eventual co-infections and transovarial transmission. A real-time qPCR assay targeting the 16S rRNA gene of N. mikurensis and other Anaplasmataceae was applied on 1356 Ixodes ricinus (I. ricinus) ticks collected from 5 sites in southern Sweden. Positive samples were subjected to Sanger sequencing. A. phagocytophilum occurred in 4.64 % of the ticks, N. mikurensis occurred in 1.33 % of the ticks and also Rickettsia was found to occur in 6.27 % of the ticks. No co-infection was detected. Some samples of tick larvae showed positive results after qPCR, indicating transovarial transmission, but none of the sequences were readable.

Ixodes genties erkės yra krauju mintantys nariuotakojai, visų – stuburinių gyvūnų, taip pat žmogaus parazitai. Ixodes ricinus erkės perneša daugybę ligų sukėlėjų, tokių kaip: erkinio encefalito (erkinio encefalito virusas), Laimo ligos (Borrelia burgdorferi sensu lato bakterijos), bebeziozės sukėlėjus (Babesia microti ir Babesia divergens). Taip pat platina ir Anaplasma phagocytophilum bakterijas. Šiuo metu erkių platinamos ligos yra dažniausios vektorių sukeliamos infekcijos Europoje. Pastaraisiais metais išpopuliarėjo molekulinės biologijos metodai, paremti patogenų nukleino rūgščių polimerazės grandinine reakcija ir RLB hibridizacija. Šie metodai laikomi tinkamiausiais, jautriausiais ir specifiškiausiais erkių platinamų ligų sukėlėjų diagnostikai. Šiame darbe naudoti molekuliniai metodai erkių platinamų ligų sukėlėjų: erkinio encefalito viruso, Anaplasma phagocytophilum, Borrelia burgdorferi sensu lato ir Babesia spp., nustatymui iš 2200 erkių, surinktų keturiuose Lietuvos rajonuose. Tyrimų metu nustatyti šeši erkinio encefalito viruso infekcijų atvejai, iš kurių penki teigiami mėginiai nustatyti Radviliškio raj. surinktose erkėse ir vienas – Utenos raj. Anaplasma phagocytophilum bakterijų infekcijos yra dažniausiai pasitaikančios infekcijos Lietuvos erkėse. Tyrimų metu nustatyti 37 (3 %) teigiami Anaplasma phagocytophilum mėginiai, iš kurių 17 (46 %) nustatyti Kėdainių raj. surinktose erkėse, 15 (32 %) – Klaipėdos, 5 (14 %) – Radviliškio ir 3 (8 %) – Utenos raj. Tarp šių... [toliau žr. visą tekstą] / Ticks of genus Ixodes that infect livestock, deer, dogs, and a wide variety of other species including humans. Ixodes ricinus can also transmit numerous diseases including tick-borne encephalitis, Lyme diseases (Borrelia burgdorferi sensu lato infections), babesiosis (Babesia microti and Babesia divergens infections). It can also spread Anaplasma phagocytophilum bacteria. Now tick-borne infections are the most frequent human vector-borne infections in Europe, the incidence of these infections has been on rise, and new infections have emerged. In recent years, molecular detection methods based on PCR amplification of the nucleic acids of pathogens and Reverse Line blot hybridization have been showed to be effective, sensitive and specific methods for diagnosis of tick-borne diseases. In this work were used molecular detection methods for tick-borne pathogens: tick-borne encephalitis virus (TBEV), Anaplasma phagocytophilum, Borrelia burgdorferi sensu lato, and Babesia spp., diagnostics in 2200 ticks collected in four Lithuanian regions forests. During investigation were identified six cases of TBEV, which five positive samples were from ticks collected in Radviliškis region and one from Utenos region forest. Anaplasma phagocytophilum bacteria infections were most frequent in Lithuanian ticks, during researches were identified 37 (3 %) cases (17 (46 %) in Kėdainių region, 15 (32 %) – Klaipėdos, 5 (14 %) – Radviliškio, and 3 (8 %) – Utenos region). In these 81 % were in adult... [to full text]