Development of a latex agglutination test with recombinant variant surface glycoprotein for serodiagnosis of surra.

Serodiagnosis of surra is commonly performed with the CATT/Trypanosoma evansi direct agglutination test. This antibody detection test is based on lyophilised bloodstream form trypanosomes propagated in rats and presenting the predominant variant surface glycoprotein (VSG) RoTat 1.2 on their surface. Recently, the N-terminal fragment of VSG RoTat 1.2 has been expressed as a recombinant protein in the yeast Pichia pastoris and showed diagnostic potential in ELISA. This recombinant antigen has now been incorporated in a latex agglutination test, the rLATEX/T. evansi. In this study, we compared the diagnostic accuracy of rLATEX/T. evansi and CATT/T. evansi with immune trypanolysis (TL) as reference test on a total of 1717 sera from camels, horses, bovines, water buffaloes, dogs and sheep. The rLATEX/T. evansi displayed a slightly better agreement with TL than CATT/T. evansi (kappa [κ] respectively 0.84 and 0.72). The sensitivities of rLATEX/T. evansi (84.2%, 95% CI 80.8-87.1) and CATT/T. evansi (84.0%, 95% CI 80.6-87.0) were similar, but rLATEX/T. evansi was significantly more specific (97.7%, 95% CI 96.7-98.4) than CATT/T. evansi (89.4%; 95% CI 87.6-91.1). We consider the rLATEX/T. evansi an alternative for the CATT/T. evansi, with the advantage that the use of a purified recombinant antigen leads to a more standardised diagnostic test with an improved specificity. Moreover, it eliminates the use of laboratory animals and can be easily scaled-up, e.g. in biofermentors.

Recombinant expression of trypanosome surface glycoproteins in Pichia pastoris for the diagnosis of Trypanosoma evansi infection.

Serodiagnosis of surra, which causes vast economic losses in livestock, is still based on native antigens purified from bloodstream form Trypanosoma (T.) evansi grown in rodents. To avoid the use of laboratory rodents in antigen preparation we expressed fragments of the invariant surface glycoprotein (ISG) 75, cloned from T. brucei gambiense cDNA, and the variant surface glycoprotein (VSG) RoTat 1.2, cloned from T. evansi gDNA, recombinantly in Pichia (P.) pastoris. The M5 strain of this yeast has an engineered N-glycosylation pathway resulting in homogenous Man5GlcNAc2 N-glycosylation which resembles the predominant Man9-5GlcNAc2 oligomannose structures in T. brucei. The secreted recombinant antigens were affinity purified with yields of up to 10mg and 20mg per liter cell culture of rISG 7529-465-E and rRoTat 1.223-385-H respectively. In ELISA, both recombinant proteins discriminated between pre-immune and immune serum samples of 25 goats experimentally infected with T. evansi. The diagnostic potential of rRoTat 1.223-385-H but not of rISG 7529-465-E was confirmed with sera of naturally infected and control dromedary camels. The results suggest that rRoTat 1.223-385-H expressed in P. pastoris requires further evaluation before it could replace native RoTat 1.2 VSG for serodiagnosis of surra, thus eliminating the use of laboratory animals for antigen production.

Characterization of Trypanosoma brucei gambiense variant surface glycoprotein LiTat 1.5.

At present, all available diagnostic antibody detection tests for Trypanosoma brucei gambiense human African trypanosomiasis are based on predominant variant surface glycoproteins (VSGs), such as VSG LiTat 1.5. During investigations aiming at replacement of the native VSGs by recombinant proteins or synthetic peptides, the sequence of VSG LiTat 1.5 was derived from cDNA and direct N-terminal amino acid sequencing. Characterization of the VSG based on cysteine distribution in the amino acid sequence revealed an unusual cysteine pattern identical to that of VSG Kinu 1 of T. b. brucei. Even though both VSGs lack the third of four conserved cysteines typical for type A N-terminal domains, they can be classified as type A.

Importancia de la terapia básica pre-tratamiento ortodóncico / The importance of pre-orthodontic treatment basic therapy

Introducción: la capacidad de los microorganismos para formar y mantenerse en el biofilm tiene un alto impacto en las infecciones crónicas, pues el mismo protege y nutre a comunidades de microorganismos que pueden influir en el tratamiento ortodóncico, aumentando la incidencia de caries y enfermedad periodontal. Objetivo: comparar la condición periodontal inicial y la alcanzada luego de la terapia básica periodontal de los pacientes que concurren a la consulta para iniciar un tratamiento ortodóncico. Materiales y métodos: se evaluaron 10 pacientes entre 14 y 30 años que concurrieron al servicio de la Cátedra de Ortodoncia de la Facultad de Odontología de la UBA. Se tomaron el índice de placa de Silness y Loe y gingival de Loe y Silness, la profundidad de sondaje y hemorragia gingival al sondaje en todas las piezas dentarias presentes en la boca del paciente. De la zona de los primeros premolares superiores se eliminó la placa supragingival y se tomaron muestras subgingivales, las cuales fueron colocadas en solución fisiológica y medio de transporte VMGAIII. Simultáneamente se realizaron extendidos del materiales y se coloreó con la técnica de Gram y de Giemsa. Luego de que los pacientes recibieran enseñanza de técnicas de higiene bucal y/o terapia básica periodontal, se volvieron a registrar dichos índices. Resultados: el índice de placa inicial presentó una mediana de 2.5 y post terapia básica, una mediana de 1.0 con una diferencia estadísticamente significativa (p=0.005) según Wilcoxon Signed Rank Test. El indice gingival inicial presentó una mediana de 2.0 y post terapia básica fue de 1.0 con una diferencia estadísticamente significativa (p<0.005 Wilcoxon Signed Test). La profundidad de sondaje pretratamiento presentó una mediana de 3.0mm con sangrado al sondaje positivo y microbiota compatible con gingivitis y periodontitis leve. Luego de enseñanza de técnicas de higiene bucal la misma fue de 1 mm con sangrado al sondaje negativo.

Structural aspects and immunolocalization of the F420-reducing and non-F420-reducing hydrogenases from Methanobacterium thermoautotrophicum Marburg.

The F420-reducing hydrogenase and the non-F420-reducing hydrogenase (EC 1.12.99.1.) were isolated from a crude extract of Methanobacterium thermoautotrophicum Marburg. Electron microscopy of the negatively stained F420-reducing hydrogenase revealed that the enzyme is a complex with a diameter of 15.6 nm. It consists of two ring-like, stacked, parallel layers each composed of three major protein masses arranged in rotational symmetry. Each of these masses appeared to be subdivided into smaller protein masses. Electron microscopy of negatively stained samples taken from intermediate steps of the purification process revealed the presence of enzyme particles bound to inside-out membrane vesicles. Linker particles of 10 to 20 kDa which mediate the attachment of the hydrogenase to the cytoplasmic membrane were seen. Immunogold labelling confirmed that the F420-reducing hydrogenase is a membrane-bound enzyme. Electron microscopy of the negatively stained purified non-F420-reducing hydrogenase revealed that the enzyme is composed of three subunits exhibiting different diameters (5, 4, and 2 to 3 nm). According to immunogold labelling experiments, approximately 70% of the non-F420-reducing hydrogenase protein molecules were located at the cell periphery; the remaining 30% were cytoplasmic. No linker particles were observed for this enzyme.