ABSTRACT
Recent studies have reported on the importance of RBCs in fish responses to viral infections and DNA vaccines. Surface-displaying recombinant bacterins (spinycterins) are a safe and adaptable prototype for viral vaccination of fish and represent an alternative method of aquaculture prophylaxis, since have been reported to enhance fish immune response. We evaluated the innate immune response of rainbow trout (Oncorhynchus mykiss) red blood cells (RBCs), head kidney, and spleen to spinycterins expressing a fragment of the glycoprotein G of viral haemorrhagic septicemia virus (VHSV), one of the most devastating world-wide diseases in farmed salmonids. We first selected an immunorelevant downsized viral fragment of VHSV glycoprotein G (frg16252-450). Then, spinycterins expressing frg16252-450 fused to Nmistic anchor-motif (Nmistic+frg16252-450) were compared to spinycterins expressing frg16252-450 internally without the anchor motif. Nmistic+frg16252-450 spinycterins showed increased attachment to RBCs in vitro and modulated the expression of interferon- and antigen presentation-related genes in RBCs in vitro and in vivo, after intravenous injection. In contrast, the head kidney and spleen of fish injected with frg16252-450, but not Nmistic+frg16252-450, spinycterins demonstrated upregulation of interferon and antigen-presenting genes. Intravenous injection of Nmistic+frg16252-450 spinycterins resulted in a higher innate immune response in RBCs while frg16252-450 spinycterins increased the immune response in head kidney and spleen. Although more studies are required to evaluate the practicality of using spinycterins as fish viral vaccines, these results highlight the important contribution of RBCs to the fish innate immune response to antiviral prophylactics.
ABSTRACT
Canonical discriminant analysis accurately separated males of Lutzomyia yencanensis (n = 36) (Ortiz), Lutzomyia cayennensis cayennensis (n = 24) (Floch and Abonnenc) and Lutzomyia micropyga (n = 28) (Mangabeira). Only seven (length of head, interocular distance, wing vein delta, genital filaments, coxite, paramere, and lateral lobe) of 20 possible morphometric measurements were required for differentiation, with a highly significant canonical correlation. The discriminant equations provided a simple, fast, and widely available statistical tool to identify accurately unknown sand fly specimens as L. yencanensis, L. c. cayennensis, or L. micropyga.
Subject(s)
Psychodidae/classification , Animals , Discriminant Analysis , Geography , Multivariate Analysis , Psychodidae/anatomy & histology , Reproducibility of Results , VenezuelaABSTRACT
The activity of engineered, peptide-displaying enzymes is modulated by binding to specific anti-peptide antibodies. This new concept of a quantitative antibody detection system allows test kits to be set up for fast diagnosis of infectious diseases. To develop a quick and homogeneous assay for the detection of human immunodeficiency virus (HIV) infection, we have explored two acceptor sites of the bacterial Escherichia coli beta-galactosidase for the accommodation of HIV antigenic peptides. Two overlapping epitopes (namely P1 and P2) from the gp41 envelope glycoprotein, contained in different sized peptides, were inserted in the vicinity of the enzyme active site to generate a set of hybrid, enzymatically active beta-galactosidases. Regulable enzymes of different responsiveness to monoclonal antibody binding were generated with both acceptor sites tested. These biosensors were also sensitive to immune sera from HIV-infected patients. Modeling data provide insight into the structural modifications in the vicinity of the active site induced by peptide insertion that strongly affect the responsiveness of the engineered proteins through different parameters of their catalytic properties.
Subject(s)
Biosensing Techniques/methods , HIV Antibodies/blood , HIV Envelope Protein gp41 , HIV Infections/diagnosis , HIV-1/immunology , beta-Galactosidase , Amino Acid Sequence , Catalytic Domain , Enzyme Stability , Epitopes , Escherichia coli/enzymology , HIV Envelope Protein gp41/genetics , Humans , Models, Molecular , Molecular Sequence Data , Peptide Fragments/chemistry , Peptide Fragments/genetics , Protein Engineering , Protein Structure, Quaternary , Protein Structure, Secondary , Recombinant Fusion Proteins , beta-Galactosidase/geneticsABSTRACT
The therapeutic effect of the emetine hydrochloride alkaloid administered intralesionally was compared with that of standard parenteral treatment with Glucantime in outbred male hamsters experimentally infected with 4 x 10(3) amastigotes of Leishmania (Viannia) braziliensis. Both chemotherapeutic agents reduced significantly (P < 0.01) the average lesion sizes in experimental animals in comparison with those untreated. The alkaloid infiltration was found to be as effective as the antimonial injection for clinical resolution. The ultrastructural effects on the Leishmania parasites exposed to emetine were observed mainly in the inner cytoplasm, which appeared disorganized, pycnotic and with loss of morphological definition; however, any known emetine hydrochloride action mechanism factor could not be directly related with ultrastructure effects detected on leishmanial parasites. Smears, conventional histopathology, culture in NNN medium and indirect immunoperoxidase method showed viable amastigotes in nodules and/or scars of all the evaluated hamsters 75 to 230 days after the end of treatment. These findings suggest that measurement of the size of cutaneous leishmania lesions does not appear to be a valid criterion for evaluating the efficiency of chemotherapy in experimental LT. Detection of leishmania parasites in the lesion scars, supports the hypothesis that man could be considered as an domestic reservoir.
Subject(s)
Antiprotozoal Agents/therapeutic use , Cicatrix/parasitology , Emetine/therapeutic use , Leishmania braziliensis/drug effects , Leishmaniasis, Cutaneous/drug therapy , Animals , Antiprotozoal Agents/administration & dosage , Antiprotozoal Agents/pharmacology , Cricetinae , Cytoplasm/drug effects , Cytoplasm/ultrastructure , Disease Reservoirs , Drug Evaluation, Preclinical , Emetine/administration & dosage , Emetine/pharmacology , Immunoenzyme Techniques , Injections, Intralesional , Leishmania braziliensis/growth & development , Leishmania braziliensis/isolation & purification , Leishmania braziliensis/ultrastructure , Leishmaniasis, Cutaneous/parasitology , Leishmaniasis, Cutaneous/pathology , Male , Meglumine/administration & dosage , Meglumine/pharmacology , Meglumine/therapeutic use , Meglumine Antimoniate , Mesocricetus , Microscopy, Electron , Organometallic Compounds/administration & dosage , Organometallic Compounds/pharmacology , Organometallic Compounds/therapeutic use , Vacuoles/drug effects , Vacuoles/ultrastructureABSTRACT
Escherichia coli lacZ is a frequently employed reporter gene for the monitoring of gene expression and recombinant protein production due the simple determination of beta-galactosidase activity in both qualitative and quantitative assays. In the absence of either total or recombinant protein synthesis, we observed a lack of correlation between protein amount and enzymatic activity in both engineered and native beta-galactosidases in Escherichia coli cells. A delayed fading of beta-galactosidase activity compared with the rapid degradation of intact protein suggests a progressive increase in enzyme-specific activity during the life of the protein. This intriguing event does not involve solubilization from major protein aggregates and it occurs both in vivo and in cell extracts, but not in solutions of purified protein. Possible explanations for this activation are examined in the context of the assisted protein folding network and proteolytic degradation of misfolded proteins.
Subject(s)
Escherichia coli/enzymology , beta-Galactosidase/metabolism , Cell Extracts , Chloramphenicol/pharmacology , Endopeptidases/metabolism , Enzyme Activation , Enzyme Stability , Escherichia coli/cytology , Escherichia coli/genetics , Genetic Vectors/genetics , Lac Operon/genetics , Protein Folding , Recombinant Proteins/biosynthesis , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Solutions , beta-Galactosidase/biosynthesis , beta-Galactosidase/genetics , beta-Galactosidase/isolation & purificationABSTRACT
Multivariate discriminant analysis was employed to discriminate on a morphological basis females of 3 closely related sand fly species, Lutzomyia townsendi (Ortiz), L. spinicrassa Morales, Osorno-Mesa, Osorno & Hoyos, and L. youngi Feliciangeli & Murillo. Principal component and canonical discriminant analysis compared a set of 31 morphological characters measured from known specimens to detect differences among the 3 species. A subset of 6 characters separated the 3 species with a high level of confidence. A simple method is presented to identify an unknown specimen as L. townsendi, L. spinicrassa, or L. youngi using these 6 morphological characters.
Subject(s)
Psychodidae/classification , Animals , Female , Multivariate Analysis , Psychodidae/anatomy & histologyABSTRACT
An entomological survey revealed that the distribution of the Lutzomyia species in Merida, Venezuela, varies with altitude. Of the 21 species encountered, 12 (57%) are known to be anthropophilic. The significance of the predominant man-biting species at any altitude is discussed in terms of the risk of transmission of cutaneous leishmaniasis. The general parous rate (and perhaps the ability of a female sandfly to survive oviposition and transmit Leishmania parasites during a second or subsequent bloodmeal) was found to be related to rainfall and altitude. The finding of Leishmania infections in wild-caught parous females of anthropophilic species indicates the high risk of acquiring leishmaniasis in the study area. Observations on the feeding habits of the sandflies indicate that some species are opportunistic feeders, attracted to a variety of hosts, including man.
Subject(s)
Leishmaniasis, Cutaneous/epidemiology , Psychodidae/physiology , Altitude , Animals , Feeding Behavior , Female , Leishmania/isolation & purification , Leishmaniasis, Cutaneous/transmission , Psychodidae/parasitology , Seasons , Time Factors , Venezuela/epidemiologyABSTRACT
The comparison among specimens of Rhodnius prolixus, at different stages of development, exposed to infections by Trypanosoma rangeli and Trypanosoma cruzi, revealed that the bugs exposed to T.cruzi showed a significant higher susceptibility to infection than that observed in the groups to T.rangeli or to the mixture of both parasites. The similar mortality rate recorded in uninfected and T.cruzi infected bugs confirms that this parasite is hermless to R.prolixus. On the other hand, T.rengeli produces its pathological effect although existence of a direct relation between the amount of ingested parasites and the mortality produced. In mixed infetion, T.cruzi does not inhibit the harmful effect of T.rangeli on R.prolixus. The life cycle of R.prolixus is not affected by high infection of T.cruzi or low infection by T.rangeli in mixed infections, the time to reach the adult stage in relation to controls, was significantly longer. The epidemiological significance of the present results, is discussed
Subject(s)
Rhodnius/parasitology , Trypanosoma cruzi/microbiology , Trypanosoma cruzi/parasitology , Trypanosoma cruzi/pathogenicityABSTRACT
Como parte de un estudio epidemiológico sobre leishmaniasis en el Estado Mérida, Venezuela, se presenta la diversidad y dispersión de las especies flebotominas identificadas en 15 localidades ubicadas en 3 pisos altitudinales entre los 175 y los 1.960 m.s.n.m. De 7.126 espécimes capturados (5.132 hombres y 1.994 femenino) se registran 24 especies de Lutzomyia, reconociéndose 10 de las llmadas antropofílicas. Se detalla la distribución de las especies en cada piso altitudinal y en el domicilio humano, el peridomicilio y el ambiente silvestre. Se discute el posible papel que juegan las diferentes especies en la transmisión de la leishmaniasis tegumentaria en cada piso altitudinal en particular y en la región andino-venezolana, en general
Subject(s)
Animals , Altitude , Leishmaniasis/transmission , Phlebotomus/physiologyABSTRACT
As part of an epidemiological study on leishmaniasis in Merida, Venezuela, the diversity and dispersion of sandflies species found in 15 localities between 175 m and 1,960 m.a.s.l., are presented. From 7,126 collected sandflies (5,132 female and 1,994 male), 24 species were identified, 10 of them recognized as anthropophilic. The relation species-altitude is presented, and the species composition found in human dwellings, periodomestic and sylvatic areas, are recorded. The possible role of the identified species on the transmission of leishmaniasis in the andean region, is discussed.
Subject(s)
Altitude , Leishmaniasis/transmission , Phlebotomus/physiology , Animals , Humans , Phlebotomus/classification , VenezuelaABSTRACT
Observaçöes individuais sobre diferentes estágios de Rhodnius prolixus expostos ao Trypanosoma rangeli, revelaram uma maior susceptilidade à infecçäo nos espécimens expostos durante os dois primeiros estádios. A taxa de mortalidade nos triatomíneos infectados foi significativamente maior que nos controles, indicando que o parasito foi responsável pelo maior número das mortes. Apresenta-se uma análise da distribuiçäo da mortalidade por estádio. A análise estatística das mortes ocorridas entre os diferentes estádios infectados, indica que o T. rangeli produz seu efeito patológico em qualquer estágio de R. prolixus, independentemente da sua susceptibilidade ao parasito. A sobrevida até adulto decresceu em todos os estádios dos triatomíneos infectados. Excetuando-se os espécimes expostos no terceiro estádio, quando foram comparados com os controles, observou-se nos triatomíneos infectados um significativo retardo para alcançar o estádio adulto. Discute-se o significado epidemiológico dos presente resultados
Subject(s)
Mice , Animals , Trypanosoma/pathogenicity , Chagas Disease/parasitology , Rhodnius/parasitologyABSTRACT
Frequent individual observations od different stages of Rhodnius prolixus exposed to Trypanosoma rangeli, revealed a higher susceptibility to infection in the bugs exposed during the two first instars. The mortality rate in infected bugs was significantly higher than in controls, indicating that the parasite was responsible for the majority of deaths. An analysis of the mortality distribution, per instar, is presented. Statistical analysis of deaths among the different infected instars, showed that T. rangeli produces its pathological effect in any stage of R. prolixus independently of its susceptibility to the parasite. The survival to adult decreased in all the infected instar bugs. A significant longer time to reach the adult stage was observed in the infected bugs when compared with controls, excepting for specimens exposed in the third instar. The epidemiological significance of the present results is discussed.
