Bacterial identification using PCR-DGGE in milk of mastitic cows from a herd of crossbreed GYR-Holstein
Municipio Obispo Ramos de Lora, Merida. Venezuela
Keywords:
bacteria, mastitis, negative culture, PCR-DGGEAbstract
In order to characterize the composition of bacteria in samples of milk from crossbreed Gyr–Holstein cows affected with mastitis, from a herd with a history of clinical mastitis from six months ago and low milk production, a culture-independent PCR - DGGE technique using as a marker the V3 region 16S rDNA was carried out. The assessment of sub clinical mastitis was performed by CMT. Samples were taken from cows that had a least 4 weeks without antibiotic therapy, and aliquots were used for bacteriology and molecular identification. Only culture negative samples were selected for PCR - DGGE. The level of sub clinical mastitis by CMT at the moment of sampling was 48.07 % (225 quarter). PCR-DGGE, revealed the presence of several types of bacteria in the samples studied, which were more closely related to bacteria of the Firmicutes and Proteobacteria phylum, which highlight the presence of the genus Acinetobacter. Most of these microorganisms are involved in antibiotic multi-resistance, so the technique becomes a useful tool for analyzing these microbial populations in cows with clinical mastitis and negative culture and establish better recommendations for control and prevention of this pathology in dairy farms. Others researches are needed to determine the implications of microorganisms isolated in this work over animal and human health.
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References
Dingwell, R., Leslie, K., Schukken, Y., Sargeant, J y Timms, L (2003). “Evaluation of the California mastitis test to detect an intramammary infection with a major pathogen in early lactation dairy cows”. The Canadian Veterinary Journal, 44(5), 413.
Dmitriev, A., Bhide, M., y Mikula, I. (2006). “cpn60 gene-based multiplex-PCR assay for simultaneous identification of Streptococcal species”. Acta Veterinaria Brno, 75(2), 235-240.
Farías Reyes, José., García Urdaneta, A., D’pool, G., Valero Leal, K., Allara, M y Angelosante, G. (2005). “Detección de mastitis subclínica en bovinos mestizos doble propósito ordeñados en forma manual o mecánica. Comparación de tres pruebas diagnósticas”. Revista Científica, 15(002).
Ferraro, L., Scaramelli, A., Troya, H.(1999) “Prevalencia de la mastitis sub-clínica bovina en Venezuela y evaluación de la prueba de mastitis de California (CMT) como prueba diagnóstica”. Revista Científica. 9, (2), 81-90.
Graber, H. U., Casey, M. G., Naskova, J., Steiner, A., y Schaeren, W. (2007). “Development of a highly sensitive and specific assay to detect Staphylococcus aureus in bovine mastitic milk”. Journal of dairy science, 90(10), 4661-4669.
Kuang, Y., Tani, K., Synnott, A. J., Ohshima, K., Higuchi, H., Nagahata, H y Tanji, Ya. (2009). “Characterization of bacterial population of raw milk from bovine mastitis by culture-independent PCR–DGGE method”. Biochemical Engineering Journal, 45(1), 76-81.
Kuehn, J, Gorden, P., Munro, D., Rong, R., Dong, Q., Plummer, P y Phillips, G. (2013) “Bacterial community profiling of milk samples as a means to understand culture-negative bovine clinical mastitis”. PloS one, 8(4), e61959.
Klostermann, K., Crispie, F., Flynn, Ja., Ross, P., Hill, C y Meaney, W. (2008). “Intramammary infusion of a live culture of Lactococcus lactis for treatment of bovine mastitis: comparison with antibiotic treatment in field trials”. Journal of dairy research, 75(03), 365-373.
Lockhart, S, Abramson, M., Beekmann, S., Gallagher, G., Riedel, St., Diekema, Daniel y Doern, G. (2007). “Antimicrobial resistance among Gram-negative bacilli causing infections in intensive care unit patients in the United States between 1993 and 2004”. Journal of clinical microbiology, 45(10), 3352-3359.
Melchior, M. B., Vaarkamp, H., y Fink-Gremmels, J. (2006). “Biofilms: a role in recurrent mastitis infections? The Veterinary Journal, 171(3), 398-407.
Muyzer, G, De Waal, E y Uitterlinden, A. (1993). Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA”. Applied and environmental microbiology, 59(3), 695-700.
Nam, H. M., Kim, J. M., Lim, S. K., Jang, K. C., y Jung, S. C . (2009). “Prevalence and antimicrobial susceptibility of gram-negative bacteria isolated from bovine mastitis between 2003 and 2008 in Korea”. Journal of dairy science, 92(5), 2020-2026.
Nam, H. M., Kim, J. M., Lim, S. K., Jang, K. C., y Jung, S. C. (2010). “Infectious aetiologies of mastitis on Korean dairy farms during 2008”. Research in veterinary science, 88(3), 372-374.
Olson, M., Ceri, H., Morck, D., Buret, A y Read, R. (2002). “Biofilm bacteria: formation and comparative susceptibility to antibiotics”. Canadian Journal of Veterinary Research, 66(2), 86.
Rea, M., Cogan, T. M., Y Tobin, S. (1992). “Incidence of pathogenic bacteria in raw milk in Ireland”. Journal of Applied Bacteriology, 73(4), 331-336.
Rebhun, W., Guard, Ch y Richards, C. (1995). Diseases of dairy cattle. Williams and Wilkins.
Scaramelli, A y González, Z. (2005). Epizootiología y diagnóstico de la mastitis bovina. Disponible desde Internet en: http://avpa. ula. ve/docuPDFs/libros_online/manualganadería/seccion5/articulo 9_s5. pdf (con acceso 20/07/09).[Links].
Sol, J., Sampimon, O. C., Snoep, J. J., y Schukken, Y. H. (1997). “Factors associated with bacteriological cure during lactation after therapy for subclinical mastitis caused by Staphylococcus aureus”. Journal of dairy science, 80 (11), 2803-2808.
Suniaga, J., Rojas, G., Hernández, J., Caamaño, J., Urbina, A. y Tovar, L. (2009). “Estudio de prevalencia de mastitis subclínica en la zona alta del estado Mérida”. Universidad de Los Andes (Venezuela). Instituto de Investigaciones Agropecuarias.
Watts, J. L.(1988). “Etiological agents of bovine mastitis. Veterinary microbiology”, 16(1), 41-66.
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