ABSTRACT
The characterization of Rhipicephalus microplus tick physiology can support efforts to develop and improve the efficiency of control methods. A sequence containing a domain with similarity to one derived from the aspartic peptidase family was isolated from the midgut of engorged female R. microplus. The lack of the second catalytic aspartic acid residue suggest that it may be a pseudo-aspartic peptidase, and it was named RmPAP. In this work we confirm the lack of proteolytic activity of RmPAP and investigate it's non-proteolytic interaction with bovine hemoglobin by Surface Plasmon Resonance and phage display. Moreover we carried out RNAi interference and artificial feeding of ticks with anti-RmPAP antibodies to assess it's possible biological role, although no changes were observed in the biological parameters evaluated. Overall, we hypothesize that RmPAP may act as a carrier of hemoglobin/heme between the tick midgut and the ovaries.
Subject(s)
Arthropod Proteins/metabolism , Aspartic Acid Proteases/metabolism , Digestive System/enzymology , Rhipicephalus/enzymology , Amino Acid Sequence , Animals , Arthropod Proteins/genetics , Arthropod Proteins/isolation & purification , Aspartic Acid Proteases/genetics , Aspartic Acid Proteases/isolation & purification , Cattle/parasitology , Cloning, Molecular , Female , Gene Expression Regulation, Enzymologic , Pseudogenes/genetics , RNA Interference , Rhipicephalus/genetics , Rhipicephalus/physiology , Sequence Homology, Amino Acid , Tick Infestations/parasitologyABSTRACT
A doença granulomatosa sistêmica associada ao consumo de Vicia villosa (Leg. Papilionoideae) foi diagnosticada em 5 bovinos no período de 2005 a 2008. Os bovinos apresentavam alopecia, lesões crostosas na pele, prurido, febre, queda da produção leiteira, anorexia e emagrecimento. O curso clínico médio da doença foi de 2 semanas. Dos bovinos analisados três morreram e dois foram eutanasiados. As lesões macroscópicas de alopecia e crostas na pele eram localizadas principalmente na face e pescoço. Observava-se nódulos multifocais a coalescentes branco-acinzentados que infiltravam vários órgãos especialmente em linfonodos, rins e coração. As lesões microscópicas consistiam na infiltração de linfócitos, macrófagos, células epitelioides, células gigantes multinucleadas, eosinófilos e plasmócitos. Linfonodos, rins, adrenal, baço e fígado de todos os bovinos apresentaram infiltrado granulomatoso, porém de intensidade variável. Nos testes imuno-histoquímicos dos órgãos com infiltrado inflamatório, as principais células visualizadas foram os linfócitos T, seguidos de macrófagos/células epitelioides/células gigantes multi-nucleadas e os linfócitos B foram raramente detectados nos locais de inflamação granulomatosa. O número reduzido de células marcadas por Ki-67 nas lesões granulomatosas, tende a indicar que a proliferação celular não foi responsável pela hipercelularidade das lesões e que o recrutamento de macrófagos e linfócitos para o local da inflamação provavelmente tenha sido o responsável pelo acúmulo de células no infiltrado inflamatório.
The systemic granulomatous disease associated with consumption of Vicia villosa (Leg. Papilionoideae family) has been diagnosed in 5 cattle from 2005 to 2008. Affected cattle showed alopecia, crusted lesions on the skin, had itching, fever, decreased milk yield, anorexia and wasting. Average clinical course was 2 weeks. Three cattle died and two were euthanized in extremis. The main gross changes are alopecic and crusts in the skin, mainly on the face and neck. There also were multifocal to coalescent whitish nodules that infiltrated several organs, but especially lymph nodes, kidneys and hearth. Microscopic changes consisted of infiltration with lymphocytes, macrophages, epithelioid cells, giant multinucleated cells, eosinophils, and plasmocytes. Lymph nodes, kidneys, adrenal gland, spleen and liver from affected cattle showed varying degrees of granulomatous infiltration. Immunohistochemical procedures on samples from affected organs revealed that T-lymphocytes and macrophages/epithelioid cells/giant multinucleated cells were the main components of the inflammatory infiltrates, B-lymphocytes were only rarely seen within. The reduced numbers of cells marked by Ki-67 in the granulomatous lesions would indicate that cell proliferation was not responsible for the hypercellularity in the lesions and that rather the recruitment of macrophages and lymphocytes to the site inflammation probably accounted for the building up of the local cellular inflammatory infiltrate.
Subject(s)
Animals , Crohn Disease/veterinary , T-Lymphocytes/cytology , Macrophages/cytology , Plants, Toxic/poisoningABSTRACT
Four monoclonal antibodies (mAbs) against extracts of embryo and gut tissue obtained from fully engorged Boophilus microplus were produced. The mAb BrBml reacted with different instars and tissues, the BrBm2 recognized only antigens present in gut extract and the mAbs BrBm3 and BrBm4 recognized vitellin. The effect of inoculation of these mAbs into fully engorged Boophilus microplus females was also evaluated. The mAbs BrBm1 and BrBm2 caused a decrease in oviposition of approximately 50% and 70%, respectively, and the mAbs BrBm3 and BrBm4 did not affect reproductive efficiency. This assay may be useful as a low-cost test to provide preliminary information on the possible effects of anti-tick antibodies in damaging ticks before attempting cattle vaccination experiments.
Subject(s)
Antibodies, Monoclonal/therapeutic use , Tick Control/methods , Ticks/immunology , Animals , Antibodies, Monoclonal/isolation & purification , Antibodies, Monoclonal/pharmacology , Antigens/isolation & purification , Enzyme-Linked Immunosorbent Assay , Mice , Mice, Inbred BALB C , Reproduction/drug effects , Ticks/drug effectsABSTRACT
The major components of protein extracts from the cattle tick Boophilus microplus eggs and larvae of various ages were characterized by molecular sieving chromatography, ion exchange chromatography and SDS-PAGE. The fractions analysed showed a changing chromatographic pattern development. A serum raised against the components of a fraction showing characteristics of vitellin strongly reacted in Western blots with the major peptides of extracts from eggs, larvae, gut and ovary. Comparison of patterns obtained by electrophoresis in non-denaturing PAGE, stained with Coomassie blue or with benzidine/hydrogen peroxide, revealed that the major proteins of these extracts are haemoproteins, possibly in different aggregation states or heterogeneous in composition.
