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Optimizing the fluorometric β-glucuronidase assay in ruminant milk for a more precise determination of mastitis

Published online by Cambridge University Press:  23 September 2011

Torben Larsen  and
Karen Aulrich
Torben Larsen*
Affiliation:
Dept. of Animal Health and Bioscience, Aarhus University, Blichers Allé 20, POB 50, DK-8830 Tjele, Denmark
Karen Aulrich
Affiliation:
Institute of Organic Farming, Federal Research Institute for Rural Areas, Forestry and Fisheries Trenthorst 32, D-23847 Westerau, Germany
*
*For correspondence; e-mail: Torben.Larsen@agrsci.dk
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Abstract

Activity of the enzyme β-glucuronidase (EC 3.2.1.31) is found in milk from ruminants with mastitis. However, the use of this enzymic activity as an indicator of mastitis has gained little attention possibly because of its low activity when compared with other mastitis indicators. The determination may therefore be less precise and the analytical procedure very time consuming and labour intensive. The present study optimized the fluorometric determination of the β-glucuronidase activity with respect to substrate concentration, pH, incubation time etc., validated the assay, and developed it into large scale analyses. The assay performance is satisfactory regarding precision, linearity etc., and it appears comparable to analogous fluorometric assays for mastitis indicators in milk. From a local dairy herd, 825 milk samples were analysed for potential mastitis indicators, i.e. β-glucuronidase, lactate dehydrogenase (LDH), alkaline phosphatase (AP), and N-acetyl-β-d-glucosaminidase (NAGase) activity, and for somatic cell counts (SCC) and the variables were compared. Activity of β-glucuronidase was moderately but significantly correlated to SCC (r=0·21; n=768) as well as the other mentioned variables (r=0·25–0·43; n=825). Simple indices based on β-glucuronidase and LDH or NAGase activity were tested as indicators of mastitis (SCC), but were not found to improve the diagnostic value. Future studies may further verify whether β-glucuronidase can compete with well-established indicators of mastitis in cows such as LDH or NAGase as well as determine whether β-glucuronidase activity, in combination with other indicators of mastitis, has an advantage. Nineteen milk samples from subclinical and latent cases of mastitis (individual quarters) were identified for specific pathogens (PCR method) and measured for β-glucuronidase activity. The activity was tested at four different pH levels (5·5, 6·0, 6·5 and 7·0) in order to investigate the possibility of discrimination between pathogens. However, all milk samples (strains of pathogens) had the same pH optimum for β-glucuronidase activity; this may indicate that enzymic activity from mammary tissue and leucocytes dominates over enzyme activity from bacterial cells.

Keywords

MastitismilkenzymeindicatorbiomarkerPCR
Type
Research Article
Information
Journal of Dairy Research , Volume 79 , Issue 1 , February 2012 , pp. 7 - 15
Copyright
Copyright © Proprietors of Journal of Dairy Research 2011

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References

Aulrich, K & Barth, K 2008 Intramammary infections caused by coagulase-negative staphylococci and the effect on somatic cell counts in dairy goats. Landbauforschung 58 59–64Google Scholar
Aulrich, K 2011 Comparison of PCR-methods for identification of Streptococcus and Staphylococccus spp. causing bovine mastitis. Landbauforschung 61 (in press)Google Scholar
BRENDA http://www.brenda-enzymes.info/index.php4Google Scholar
Chagunda, GGC, Larsen, T, Bjerring, M & Ingvartsen, KL 2006 L-lactate dehydrogenase and N-acetyl-β-d-glucosaminidase activities in bovine milk as indicators of non-specific mastitis. Journal of Dairy Research 73 431–440CrossRefGoogle ScholarPubMed
Couto, I, Pereira, S, Miragaia, M, Sanches, IS & de Lencastre, H 2001 Identification of clinical staphylococcal isolates from humans by internal transcribed spacer PCR. Journal of Clinical Microbiology 39 3099–3103Google Scholar
DVG 2009 [Guidelines for the extraction of milk samples under antiseptic conditions and Guidelines for the isolation and identification of mastitis] Giessen, Germany: DVG-Verlag, Giessen, pp. 91Google Scholar
Fang, W, Vikerpuur, M & Sandholm, M 1995 A fluorometric β-glucuronidase assay for analysis of bacterial growth in milk. Veterinary Microbiology 46 361–367Google Scholar
Griffiths, MW 1986 Use of milk enzymes as indices of heat treatment. Journal of Food Protection 49 696–705CrossRefGoogle ScholarPubMed
Hurley, WL 1987 Assays and activities of glycosidic enzymes in bovine peripheral blood leucocytes. Veterinary Immunology and Immunopathology 16 85–93CrossRefGoogle Scholar
Hurley, WL, McKee, TM & Cue, DR 1987 Lysosomal enzymes in bovine mammary leucocytes during the nonlactating period. Veterinary Immunology and Immunopathology 16 95–105CrossRefGoogle ScholarPubMed
Iritani, B & Inzana, TJ 1988 Evaluation of a rapid tube assay for presumptive identification of Escherichia coli from veterinary specimens. Journal of Clinical Microbiology 26 564–566Google Scholar
Kiermeier, F & Güll, J 1966 [Determination of β-glucuronidase in cow milk]. Naturwissenschaften 53 613–614CrossRefGoogle Scholar
Kiermeyer, F & Doruk, M 1972 Inactivation of β-glucuronidase in milk by heat. Zeitschrift für Lebensmittel-Untersuchung und-Forschung 150 220–224Google Scholar
Kitchen, BJ 1976 Enzymatic methods for estimation of the somatic cell count in bovine milk. Journal of Dairy Research 43 251–258Google Scholar
Jenness, R & Patton, S 1959 In: Principles of Dairy Chemistry New York, USA: John Wiley and Sons. pp. 200–202Google Scholar
Larsen, T 2005 Determination of lactate dehydrogenase (LDH) activity in milk by a fluorometric assay. Journal of Dairy Research 72 209–216Google Scholar
Larsen, T, Røntved, CM, Ingvartsen, KL, Vels, L & Bjerring, M 2010 Enzyme activity and acute phase proteins in milk utilized as indicators of acute clinical E. coli LPS-induced mastitis. Animal 4 1672–1679CrossRefGoogle Scholar
Matthews, KR, Oliver, SP & King, SH 1991 Evaluation of two fluorometric assays for identification of Streptococcus species isolated from bovine mammary glands. Journal of Dairy Science 74 421–425CrossRefGoogle Scholar
McDonald, WL, Fry, BN & Deighton, MA 2005 Identification of Streptococcus spp. causing bovine mastitis by PCR-RFLP of 16S-23S ribosomal DNA. Veterinary Microbiology 111 241–246Google Scholar
Nagahata, H, Saito, S & Noda, H 1987 Changes in N-acetyl-β-D-glucosaminidase and β-glucuronidasde activities in milk during bovine mastitis. Canadian Journal of Veterinary Research 51 126–134Google ScholarPubMed
Oliszewski, R, Nunez de Kairuz, MS, Gonzales de Elias, SN & Oliver, G 2002 Assessment of β-glucuronidase levels in goat's milk as an indicator of mastitis: Comparison with other mastitis detection methods. Journal of Food Protection 65 864–866Google Scholar
Perdigon, G, Medici, M, Cecilia, M, Nader de Macias, ME, Haedo, R, Oliver, G & Peche de Ruiz Holgado, AA 1986 Significance of the presence of bovine milk β-glucuronidase in mastitis detection. Journal of Dairy Science 69 27–31Google Scholar
Perdigon, G, Medici, MR, Oliver, G & Peche de Ruiz Holgado, AA 1988 Relationship between β-glucuronidase levels and cell counts in bulk milk. Milchwissenschaft 43 25–26Google Scholar
Sarhan, HR & Foster, HA 1991 A rapid fluorogenic method for the detection of Escherichia coli by the product of β-glucuronidase. Journal of Applied Bacteriology 70 394–400Google Scholar
Schaufuss, P, Lämmler, C & Blobel, H 1986 Rapid differentiation of Streptococci isolated from cows with mastitis. Journal of Clinical Microbiology 24 1098–1099Google Scholar