RESUMEN

Industrial crops including coconut palm and other palm species are seriously infested by red palm weevil (RPW), resulting in significant economic damage globally. Therefore, this study aimed to develop a mycoinsecticide utilizing conidia of Metarhizium anisopliae to control RPW and sought to investigate a new emulsion formulation for the influences of storage temperature and heat stress on conidia germination in an oil-in-glycerol emulsion system. The mycoinsecticide is an emulsion formulation which comprises an oil carrier, non-ionic surfactants, water, and glycerol, which was optimized by premixing the oil and non-ionic surfactant in different weight ratios (1:0, 9:1, 8:2, 7:3, 6:4, 5:5, 4: 6, 3: 7, 2:8, 1:9, and 0:1). From three selected oil-in-glycerol formulations, F25 was more stable in storage and had a smaller particle size (between 154.3 and 236.4 nm in diameter) and stable zeta potential (above + 30 mV) with low surface tension (29.83 ± 0.24 mN/m to 30.72 ± 0.11 mN/m at room temperature. Extended conidial viability was observed at 4 °C overall; the emulsion formulation maintained 12-15% conidial viability until the eighth week at room temperature. Heat of over 30 °C showed an inhibitory effect on conidial germination. This study revealed that the oil-in-glycerol formulation was stable and able to prolong conidial shelf life as compared to non-formulated conidia.

RESUMEN

AIM: The objective of this study was to produce recombinant protein GRA-4 (rGRA-4) of a local Toxoplasma gondii isolate as a candidate for a toxoplasmosis diagnosis kit in Escherichia coli BL21 (DE3) competent cells using pET SUMO plasmid. MATERIALS AND METHODS: Samples used were stock T. gondii tachyzoites DNA from the Parasitology Laboratory, Faculty of Veterinary Medicine, Gadjah Mada University, Yogyakarta. Amplified GRA-4 polymerase chain reaction product of T. gondii tachyzoite DNA was cloned in the pET-SUMO TAR cloning vector. The GRA-4 gene from T. gondii local isolate was sequenced, followed by plasmid transformation, recombinant plasmid DNA isolation, and recombinant protein expression in DE3 competent cells. RESULTS: The amplification product of GRA-4 T. gondii gene was 1036 bp, with 48 kDa molecular weight after expression in DE3 competent cells. An alignment of the amino acid sequence of GRA-4 from the local isolate which was cloned with GRA-4 was obtained from NCBI database and showed 99.61% homology to the predicted GRA-4 from the T. gondii Izatnagar isolate. Amino acid sequence of the predicted GRA-4 protein from local isolate was different at positions 19 and 304. CONCLUSION: This research cloned rGRA-4 in pET SUMO plasmid.

RESUMEN

BACKGROUND: Toxoplasmosis is a common infection all around the world. During pregnancy; it may lead to congenital disorders or abortion in human and animals. Severe damage of toxoplasmosis indicates to require effective vaccine. One of dense granules antigen is GRA4 that secrete from tachyzoite and bradyzoite. GRA4 genome is unique without intron and is one of the major immunogenic proteins from Toxoplasma gondii. METHODS: We confirmed the cloning of GRA4 gene into pcDNA3 by restriction enzyme and PCR of GRA4 gene with pcGRA4 plasmids as template. Then with using calcium-phosphate method we transfected the pcGRA4 into CHO (Chinesehamster ovary) cells. The yielded protein was separated by SDS-PAGE and moved by electroblotting to nitrocellulose paper. RESULTS: Result of SDS-PAGE analysis showed the appearance of band approximately 42 kDa which was absent in the negative control, that was able to identify toxoplasmosis antibody IgM+ serum in western blot analysis. CONCLUSION: pcGRA4 plasmid is able to synthesis of antigenic protein in CHO cells. The ability of pcGRA4 for induction of protective immune response against toxoplasmosis will be evaluated in mouse model.

RESUMEN

The development of an effective and safe vaccine to prevent Toxoplasma gondii infection is an important aim due to the great clinical and economic impact of this parasitosis. We have previously demonstrated that immunization with the serine protease inhibitor-1 (TgPI-1) confers partial protection to C3H/HeN and C57BL/6 mice. In order to improve the level of protection, in this work, we combined this novel antigen with ROP2 and/or GRA4 recombinant proteins (rTgPI-1+rROP2, rTgPI-1+rGRA4, rTgPI-1+rROP2+rGRA4) to explore the best combination against chronic toxoplasmosis in C3H/HeN mice. All tested vaccine formulations, administered following a homologous prime-boost protocol that combines intradermal and intranasal routes, conferred partial protection as measured by the reduction of brain cyst burden following oral challenge with tissue cysts of Me49 T. gondii strain. The highest level of protection was achieved by the mixture of rTgPI-1 and rROP2 proteins with an average parasite burden reduction of 50% compared to the unvaccinated control group. The vaccine-induced protective effect was related to the elicitation of systemic cellular and humoral immune responses that included antigen-specific spleen cell proliferation, the release of Th1/Th2 cytokines, and the generation of antigen-specific antibodies in serum. Additionally, mucosal immune responses were also induced, characterized by secretion of antigen-specific IgA antibodies in intestinal lavages and specific mesenteric lymph node cell proliferation. Our results demonstrate that rTgPI-1+rROP2 antigens seem a promising mixture to be combined with other immunogenic proteins in a multiantigenic vaccine formulation against toxoplasmosis.

Asunto(s)

Antígenos de Protozoos/inmunología , Vacunas Antiprotozoos/normas , Toxoplasma/inmunología , Toxoplasmosis Animal/prevención & control , Animales , Anticuerpos Antiprotozoarios/sangre , Línea Celular , Enfermedad Crónica , Citocinas/metabolismo , Femenino , Fibroblastos/parasitología , Prepucio/citología , Humanos , Inmunoglobulina A Secretora/análisis , Inmunoglobulina G/sangre , Mucosa Intestinal/inmunología , Masculino , Proteínas de la Membrana/inmunología , Ratones , Ratones Endogámicos C3H , Proteínas Protozoarias/inmunología , Bazo/citología , Bazo/inmunología , Vacunas Sintéticas/normas

RESUMEN

Objective To construct a prokaryotic expression system containing the dense granule protein 4(GRA4) of Toxoplasma gondii,purify the expressed protein and detect its immunogenicity.Methods The specific fragment of GRA4 gene was amplified by PCR.After subcloning the prokaryotic expression recombinant pET,GRA4,the expressed product was purified with His?BindTM affinity chromatography and analyzed by Western blot.BALB/c mice were immunized with the GRA4 recombinant protein,and the antibody IgG titer was detected by ELISA.Results The pET,GRA4 prokaryotic expression system was obtained.The MW of the expressed protein was Mr 40 000 and formed in inclusion body.After purification,the recombinant protein could be specifically recognized by the T.gondii infected rabbit serum.Mice immunized with the purified recombinant protein elicited high titer of IgG antibody.Conclusion The pET,GRA4 recombinant protein was successfully expressed and purified,which shows the immunogenicity.