Antigenic characterization of Trypanosoma evansi using sera from experimentally and naturally infected bovines, equines, dogs, and coatis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Toxoplasma gondii: cloning, sequencing, expression, and antigenic characterization of ROP2, GRA5 and GRA7

Toxoplasma gondii is an intracellular obligate protozoan, which infects humans and warm-blooded animals. The aim of the present study was to clone the rop2, gra5 and gra7 genes from T. gondii RH strain and to produce recombinant proteins. The rop2, gra5 and gra7 gene fragments produced by polymerase chain reaction were cloned into the pET102/D-TOPO(R) vector which contains thioredoxin and polyhistidine tags at the C-and N-ends, respectively, and is expressed in Escherichia coli BL21(DE-3). The expression fusion proteins were found almost entirely in the insoluble form in the cell lysate. These recombinant proteins were purified with an Ni-NTA column. Concentrations of the recombinant antigens produced in the E. coli BL21-star ranged from 300 to 500 mu g/mL growth media, which was used to immunize rabbits. We observed an identity ranging from 96 to 97% when nucleotide sequences were compared to GenBank database sequences. Immunocharacterization of proteins was made by indirect immunofluorescence assay. These proteins will be used for serodiagnosis and vaccination.

Enzymatic Hydrolysis of Sweet Lupin, Chickpea, and Lentil 11S Globulins Decreases their Antigenic Activity

We investigated the effects of treatments with the enzymes pepsin and trypsin on the in vitro immunological reactivity of the major globulins found in the seeds of sweet lupin, chickpea, and lentil. Polyclonal major globulin-specific antiserum was obtained by immunization of rabbits with a solution of the 11 S globulin of each legume. The globulins were hydrolyzed with pepsin and trypsin for 1, 5, 15, and 30 min. The native globulins and their hydrolysates were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting to identify the polypeptide bands with antigenic activity, and the hypoantigenicity of the hydrolysates was analyzed by enzyme-linked immunosorbent assay. Our results show that enzymatic treatment of the major storage protein (11 S globulin) of sweet lupin, chickpea, and lentil with pepsin or trypsin lead to the formation of large amounts of short peptides and free amino acids that do not allow antibody binding, resulting in a weakened immunoreactivity.

IgE antibody response to the main antigenic component of Paracoccidioides brasiliensis in patients with paracoccidioidomycosis

Paracoccidioidomycosis (PCM) has two main clinical presentations, a chronic form (CF) and an acute, more severe form (AF). The AF is associated with a more marked dysfunction of the patient's immune response, and a distinct anti-Paracoccidioides brasiliensis immunoglobulin (Ig)A and IgG antibody subclass expression, compared with that seen in the CF. In this study we investigated the presence of IgE antibodies against the main P. brasiliensis antigen (a 43-kDa molecule) in the serum of PCM patients using an enzyme-linked immunosorbent assay. We found that 100% of the AF patients (n = 16) produced IgE antibodies, mostly at high levels, whereas only 9 (27%) out of 33 CF patients produced this isotype. Interestingly, these nine patients presented higher serological titers on the counter-immunoelectrophoresis assays than did those who did not produce IgE; a finding that suggests that they had a relatively more severe disease. As IgE is a characteristic feature of the AF patients, and switching to a positive IgE response is dependent on interleukin-4, our results support the notion that the relatively more severe impairment of cellular immunity in the AF is probably related to a Th-2 pattern of immune response.

Antigenic Activity of Bacterial Endodontic Contents from Primary Root Canal Infection with Periapical Lesions against Macrophage in the Release of Interleukin-1 beta and Tumor Necrosis Factor alpha

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Extensive antigenic and genetic variation among foot-and-mouth disease type A viruses isolated from the 1994 and 1995 foci in São Paulo, Brazil

Nine foot-and-mouth disease virus (FMDV) type A isolates recovered from the field FMD foci in São Paulo State, Brazil, during 1994 and 1995 (a period preceding the last reported focus of FMD in 1996 in this state) were compared among themselves and with the reference vaccine strain A(24)Cruzeiro. The techniques used were sandwich ELISA, virus neutralization (VN), polyacrylamide gel electrophoresis (PAGE) of the structural polypeptides and direct sequencing of the VP1-coding region (1D gene). Results of VN were recorded as serological relationships R and those from ELISA were expressed as percentage of the homologous reaction r. ELISA and VN gave comparable results (correlation coefficient, 0.936) allowing assignment of these field viruses to four groups which were distinct from the A(24)Cruzeiro strain. PAGE and ID nucleotide sequencing were also able to distinguish between these viruses. The high level:of genetic and antigenic variation found when comparing the A(24)Cruzeiro vaccine strain and type A strains recovered, from the last identified foci of FMD came from a formerly endemic area where vaccination with polyvalent vaccines (O(1)Campos, A(24)Cruzciro and C(3)Indaial) had been extensively applied. The similarity between the results of the serological and genetic analyses suggest that the antigenic differences found are mainly located in the 1D protein. (C) 2002 Elsevier B.V. B.V. All rights reserved.

Antigenic typing of brazilian rabies virus samples isolated from animals and humans, 1989-2000

Amostras de vírus rábico isoladas de animais e humanos no período de 1989 a 2000 foram tipificadas antigenicamente com a utilização de um painel de anticorpos monoclonais contra a nucleoproteína viral, pré-estabelecido para o estudo da epidemiologia molecular do vírus rábico isolado nas Américas. As amostras testadas foram isoladas no laboratório de diagnóstico do Instituto Pasteur e outros centros de diagnóstico de raiva no Brasil. Além das cepas de vírus rábico fixo CVS-31/96-IP, mantida em cérebro de camundongos e a PV-BHK/97, mantida em cultura de células, cepas de vírus rábico isoladas de cães, gatos, bovinos, eqüinos, morcegos, ovinos, caprino, suínos, raposa, sagüí, coatí, guaxinim e humanos, totalizaram 330 amostras. Seis variantes antigênicas foram definidas, compatíveis com perfís observados no painel de anticorpos monoclonais pré-estabelecido utilizado, as de número 2 (cão), 3 (Desmodus rotundus), 4 (Tadarida brasiliensis), 5 (Vampiro da Venezuela), 6 (Lasiurus cinereus) e Lab (reagente a todos os anticorpos utilizados), além de outros seis perfís desconhecidos, não compatíveis com aqueles observados no painel utilizado. A maior variabilidade foi observada entre as amostras isoladas de morcegos insetívoros e a variante mais comum isolada entre as espécies foi a variante 3 (Desmodus rotundus). Estes fatos podem representar a existência de múltiplos ciclos de transmissão independentes, envolvendo diferentes espécies de morcegos.

Antigenic similarities to Paracoccidioides brasiliensis in thermo-dependent dimorphic fungi isolated from soil in Botucatu, SP, Brazil

We compared the antigenic characteristics of two thermo-dependent dimorphic fungi isolated from soil in Botucatu, an endemic area of paracoccidioidomycosis (PCM) and Paracoccidioides brasiliensis. The soil isolates grew as cerebriform colonies at 37 degrees C (yeast form) and as cottonous colonies at 25 degrees C (mycelial form). No pathogenicity for ddY mice or hamsters were observed. In immunodiffusion test, there were precipitation bands between the 2 soil isolates and pooled PCM patient sera. There were also common precipitation bands at 21, 50 and 58 kDa between the soil isolates antigens and PCM patient sera by Western-blotting, but no gp43 kDa band. No gene for gp43 kDa protein was detected in the soil isolates by PCR. The fact that these isolates were obtained from an endemic area of PCM and there were some antigenic similarities between the soil isolates and P. brasiliensis in immunodiffusion test and Western-blotting may have some importance in epidemiological surveys done with paracoccidioidin as well interfering with the immune response of the exposed population.

Localization of Antigenic Sites in Unfed Nymphs of Amblyomma triste Koch 1844 (Acari: Ixodidae) Ticks by Immunohistochemistry

P>The reactivity of sera collected from guinea pigs after three infestations with Amblyomma triste nymphs on histological sections of the same tick species was investigated through immunohistochemistry to identify potential target cells and tissues. Six guinea pigs were infested thrice, at 30 day intervals, with 30 nymphs of A. triste per animal per infestation. Blood samples were collected from the guinea pigs 15 days after each infestation for serum separation; normal serum was obtained before the first infestation as control. Unfed A. triste nymphs' histological sections were submitted to indirect immunohistochemistry technique by using normal or hyperimmune guinea pig serum as primary antibody and a goat IgG-alkaline phosphatase-APase conjugate as secondary antibody. A weak to moderate APase activity was observed in cells of salivary glands, midgut and haemolymph of unfed nymphs incubated with hyperimmune serum.

Western blot analysis of tick antigens from a Rhipicephalus sanguineus unfed larval extract and identification of antigenic sites in tick sections using immunohistochemistry. A comparative study between resistant and susceptible host species

Most parasite-host relationships are characterized by the development of resistance by the host, thus limiting the number of parasites. However, some cases are very unusual. In the relationship of the domestic dog with the brown dog-tick Rhipicephalus sanguineus this does not occur, whereas guinea pigs develop efficient resistance. Sera from domestic dogs, crab-eating foxes and guinea pigs collected before and after infestation with R. sanguineus ticks, and after immunization with a whole tick adult or larval homogenate, were used in Western blot analysis to compare and identify potential important antigens from a tick larval homogenate. The same sera were tested in an indirect immunohistochemistry assay in an attempt to compare relevant antigenic sites on histological tick sections. The immunoblotting displayed antigens recognized only by the guinea pigs, as well as several shared antigens between host species, depending on the kind of immunization. Immunohistochemistry revealed probable antigenic sites on the cells and tissues of ticks, which varied depending on the kind of immunization (infestation or vaccination) and the animal species involved.

Electrophoretic patterns of lipopolysaccharides and antigenic analysis of soybean bradyrhizobia

Several serological methods have been used for the characterization and identification of soybean bradyrhizobia. However, some problem were non-reactivity of certain strains and cross-reactivity among others. Since lipopolysaccharide (LPS) can often be used in strain identification, the objective was to investigate the antigenic properties and polyacrylamide gel electrophoretic pattern of 12 Brazilian strains of Bradyrhizobium japonicum that nodulate soybean and to compare them to standard strains. The close correlation between the LPS patterns obtained by SDS-PAGE and the serological analysis permitted us to assign the strains to nine groups different or the same as the standard strains.

Immunosensor based on immobilization of antigenic peptide NS5A-1 from HCV and silk fibroin in nanostructured films

The peptide NS5A-1 (PPLLESWKDPDYVPPWHG), derived from hepatitis C virus (HCV) NS5A protein, was immobilized into layer-by-layer (LbL) silk fibroin (SF) films. Deposition was monitored by UV-vis absorption measurements at each bilayer deposited. The interaction SF/peptide film induced secondary structure in NS5A-1 as indicated by fluorescence and circular dichroism (CD) measurements. Voltammetric sensor (SF/NS5A-1) properties were observed when the composite film was tested in the presence of anti-HCV. The peptide-silk fibroin interaction studied here showed new architectures for immunosensors based on antigenic peptides and SF as a suitable immobilization matrix. © 2013 American Chemical Society.

Leishmania (Viannia) shawi purified antigens confer protection against murine cutaneous leishmaniasis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)