An Assessment of Microbiological Methods to Test Sterility of Foot-and-mouth Disease Vaccines Produced in Brazil

Carla Rosane Rodenbusch, Luiz Roberto da Silveira, Álvaro Ricardo Bavaresco, Marcus Vinícius Burgel Sfoggia


Background: Foot-and-mouth vaccines are an important tool in the control and eradication of the disease. In order to be commercialized, vaccines produced in Brazil undergo an evaluation process by health authorities, which includes sterility testing, residual active virus, potency, thermal stability, volume and non-structural protein activity. Sterility tests described in the Brazilian Pharmacopeia and by the World Organization for Animal Health (OIE) include direct inoculation and membrane filtration methods. The objective of the present study was to evaluate these two methods used to analyze sterility of vaccines against foot-and-mouth disease produced in Brazil.

Materials, Methods & Results: Vaccines produced by the six main laboratories in Brazil were initially tested for filtration capacity. The sensitivity of the two techniques was determined artificially contaminating vaccines using known bacterial concentrations. Vaccines (9 bottles) from the same manufacturer were inoculated with 5 mL of steady-state growths of Pseudomonas aeruginosa, Candida albicans, and Clostridium sporogenes to final concentrations of 0.1, 1 and 10 CFU/ mL and a final volume of 55 mL. Bottles were manually shaken for 1 min to complete homogenization of contents. Then, 10 mL of each flask were used in assessment of the direct inoculation method, and 10 mL were used to evaluate the membrane filtration technique. Direct inoculation was carried out inoculating 1 mL of the experimentally contaminated vaccine in five tryptic soy broth (TSB) and fluid thioglycollate medium (FTM) bottles. The membrane filtration technique was carried out filtrating 10 mL of the challenged vaccines in a peristaltic pump system (SteritestTM Pump System), where vaccines were initially solubilized in Triton X-100 to promote filtration. Next, membranes are incubated in TSB and FTM. These use two types of culture medium, tryptic soy broth (TSB) and fluid thioglycollate medium (FTM), with incubation times of 20-25ºC and 30-35ºC, respectively, to detect fungi, yeasts, and aerobic and anaerobic bacteria. The medium is incubated for 14 days, to enable the detection of slow-growth microorganisms that may be in a latent stage or weakened due to the extreme conditions of the production process (like the use of cleaning and disinfection agents, ultraviolet light, and preservers, for instance). All vaccines were effectively filtered in the SteritestTM Pump System. Membrane filtration and direct inoculation presented the same sensitivity to detect yeasts (0.1 CFU/mL) and anaerobic organisms (1 CFU/mL). For the detection of aerobic organisms, membrane filtration was 100 times more sensitive, compared to direct inoculation.

Discussion: The specialized literature also reports that, apart from the higher sensitivity, membrane filtration affords to reduce contamination during the procedures, since it is carried out in a closed system. In addition, it is indicated in the analysis of large sample volumes. Moreover, membrane filtration reduces the occurrence of false positive results, since it removes the excess vaccine volume from the culture medium, which may be mistaken for turbidity caused by bacterial growth. In this sense, the membrane filtration technique is more appropriate in the control of vaccine sterility in foot-andmouth disease prevention strategies, and is an interesting tool to improve quality control of the product.


Foot-and-mouth disease; vaccine; direct inoculation; membrane filtration; sterility.

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Abaracón B. & Olascoaga R.C. 1984. Vacinas contra a febre aftosa. A Hora Veterinária. 17: 45-52.

Ministério da Agricultura Pecuária e Abastecimento. 2008. Instrução Normativa 50/2008 de 24/09/2008. Regulamento Técnico para a Produção, Controle da Qualidade, Comercialização e Emprego de Vacinas Contra a Febre Aftosa, 7p. Disponível em: . [Acessed online June 2015.]

Agência Nacional de Vigilância Sanitária. 2010. Farmacopéia Brasileira. 5.ed. v.1. Métodos biológicos, Ensaios biológicos e Microbiológicos. pp.256-260. Disponível em: . [Acessed online May 2015].

Cortés A., Sandino C. & Arias J. 2003. Validación de la prueba de esterilidad para vacunas virales preparadas em vehiculos oleoso y acuoso. Revista de la Faculdad de Farmacia. 45: 36-43.

European Commission. 2012. Guide to Good Manufacturing Practice for Medical Products for Human and Veterinary Use. Manufacture of Sterile Medicinal Products. 16p. Disponível em: . [Acessed online May 2015].

Hernandez-Lopez J., Guzman-Murillo A. & Vargas-Albores F. 1995. Quantification of pathogenic marine vibrio using membrane filter technique. Journal of Microbiology Methods. 21: 143-149.

Jamal S.M. & Belsham G.J. 2013. Foot-and-mouth disease: past, present and future. Veterinary Research. 44: 116129.

MacLachlan N.J. & Dubovi E.J. 2011. Picornaviridae. In: Fenner’s Veterinary Virology. London: Academic Press, pp.431-435.

OIE - World Organization for Animal Health. 2013. Manual of diagnostic test and vaccines for terrestrial animals. Tests for Sterility and Freedom from Contamination of Biological Materials, 10p. Disponível em: . [Acessed online June 2015].

Parveen S., Kaur S., David S.A.W., Kenney J.L., McCormick W.M. & Gupta R.K. 2011. Evaluation of growth based rapid microbiological methods for sterility testing of vaccines and other biological products. Vaccine. 9: 80128023.

Pinto T.J.A., Kaneko T.M. & Ohara M.T. 2003. Controle biológico de qualidade de produtos farmacêuticos, correlatos e cosmético. 2.ed. São Paulo: Atheneu, 325p.

Sharp J. 1995. What do we mean by sterility? PDA Journal of Pharmaceutical Science and Technology. 49: 90-92.

Von Woedtke T. & Kramer A. 2008. The limits of sterility assurance. GMS Krankenhaushygiene. Interdisziplinär. 3: 1-10.

Willems T., Lefebvre D.J., Goris N., Diev V.I., Kremenchugskaya G.P., Haas B. & De Clercq K. 2012. Characteristics of serology-based vaccine potency models for foot-and-mouth disease virus. Vaccine. 30: 5849-5855.


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