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Standardization and establishment of an in vitro culture of Blomia tropicalis toward technological development purposes

Luis Acuna Cantillo1, Gloria Garavito1, Dary L. Mendoza Meza2 and Eduardo A. Egea Bermejo1*
  • 1 Universidad del Norte, Departamento de Medicina, Colombia
  • 2 Universidad del Atlántico, Departamento de Química, Facultad de Ciencias Básicas, Colombia

The house dust mite B.tropicalis is prevalent in the tropical zone, mainly in Coast Cities. It is a risk factor for asthma and rhinitis and up today is accepted as a trigger of allergic inflammation cascade. The principal objective was standardize and establishment an In vitro culture of house dust mite B.tropicalis for obtaining protein extracts with biomedical purposes application. B.tropicalis was isolated from house dust of asthmatic patients in Barranquilla city. Dust mattress was aspirated and mixed with fish feed (Tetramin®) in 1:1 ratio in Petri dishes of 100 x 15 mm, kept for two weeks in bioclimatic chamber at average temperature of 26.5 °C (± 1,04) and average relative humidity of 74% (± 0.07). A number of 100 mites of B.tropicalis in adult stage, were transferred to Erlenmeyer flasks with 20 grams of sterile Tetramin® and grown under the same conditions of temperature and relative humidity. To prevent escape of mites, the flasks were sealed with a sterile gauze plug. Contamination of cultures with other species of mites and fungi was monitored periodically. For separation of mites from high density cultures Tullgren modified method was used. The extraction of total proteins of B.tropicalis was performed by osmotic lysis with ammonium bicarbonate and centrifugation, the supernatant was dialyzed and lyophilized. Protein concentration was determined by Bradford microassay and the electrophoretic profile by SDS-PAGE. Antigenic evaluation was performed by Dot Blot and hydrolase enzyme activity was assessed using Api Zym kit (bioMérieux®). The cultures showed a maximum growth between weeks 4 and 5 of culture initiation, as evidenced by the presence of mites in the flask gauze packing, after week 5 dead mites were observed. The average yield of mites was 5 grams per flask. The average concentration of total protein was 1162 µg/mL (± 1094.6). The electrophoretic pattern showed bands between 20 and 100 kDa in protein extracts. It was found a profile of IgE binding proteins showing a 20 bands that recognized the main allergens from the extract and the enzymatic activity was positive for acidic and alkaline phosphatase, esterase C4 and lipase, leucine arylamidase, trypsin, Naphthol ASBI phosphohydrolase, alpha and beta galactosidase, alpha and beta -glucosidase, beta glucorinidase, N-acetyl-beta-glucosaminidase and alpha-mannosidase. We present a method for the development of in vitro cultures of B.tropicalis to obtain protein extracts that could be used as tools for the diagnosis and treatment of allergic diseases in the tropic.

Acknowledgements

The authors thank the Division of research, development and innovation of the Universidad del Norte for the support given to this study.

References

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Keywords: culture, Blomia tropicalis, Allergic., Protein extracts, Electrophoretic pattern, IgE binding protein, Enzymatic activity.

Conference: IMMUNOCOLOMBIA2015 - 11th Congress of the Latin American Association of Immunology - 10o. Congreso de la Asociación Colombiana de Alergia, Asma e Inmunología, Medellin, Colombia, 13 Oct - 16 Oct, 2015.

Presentation Type: Poster Presentation

Topic: Allergy

Citation: Acuna Cantillo L, Garavito G, Mendoza Meza DL and Egea Bermejo EA (2015). Standardization and establishment of an in vitro culture of Blomia tropicalis toward technological development purposes. Front. Immunol. Conference Abstract: IMMUNOCOLOMBIA2015 - 11th Congress of the Latin American Association of Immunology - 10o. Congreso de la Asociación Colombiana de Alergia, Asma e Inmunología. doi: 10.3389/conf.fimmu.2015.05.00264

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Received: 25 May 2015; Published Online: 14 Sep 2015.

* Correspondence: MD, PhD. Eduardo A Egea Bermejo, Universidad del Norte, Departamento de Medicina, Barranquilla, Atlántico, Colombia, eegea@uninorte.edu.co