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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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