Åse Sternesjö

Haptoglobin and serum amyloid A in relation to the somatic cell count in quarter, cow composite and bulk tank milk samples

Milk somatic cell count (SCC) is the gold standard in diagnosis of subclinical mastitis, and is also an important parameter in quality programmes of dairy cooperatives. As routine SCC analysis is usually restricted to central laboratories, much effort has been invested in the search for alternative biomarkers of mastitis and milk quality, including the presence in the milk of the acute phase proteins (APP), haptoglobin (Hp) and serum amyloid A (SAA). The aim of this study was to investigate relationships between Hp, SAA and SCC in quarter, cow composite, and bulk tank milk samples. Cows (n=165), without any clinical signs of disease or abnormalities in the milk or udder, from three different dairy farms, were used. Cow composite milk samples from all cows delivering milk at the sampling occasion were taken once in each herd. In one of the farms, representative quarter milk samples (n=103) from 26 cows were also collected. In addition, bulk tank milk samples from 96 dairy farms were included in the study. Samples were analysed for Hp, SAA and SCC, and relationships between the parameters were evaluated at quarter, cow and tank milk levels using Chi-square analysis. Milk samples were categorized according to their SCC, and the presence, or no presence, of SAA and Hp, based on the detection limits of the screening methods (0.3 mg/l and 1.0 mg/l for SAA and Hp, respectively). Hp and SAA were found in milk at quarter, cow composite and bulk tank levels. A large proportion (53%) of the animals had detectable milk concentrations of APP, and SAA was detected more frequently, and at higher concentrations than Hp, regardless of sample type. SAA was detected in as many as 82% of the bulk tank milk samples. Significant relationships were found between Hp, SAA and SCC at quarter and cow composite milk levels, but only between SAA and SCC at bulk tank milk level. Detectable levels of APP were more common at high SCC.

Biosensor assay for determination of haptoglobin in bovine milk

Despite more than 30 years of research into mastitis diagnostics, there are few alternatives to the somatic cell count (SCC) in practical use for identification of cows with subclinical mastitis. Mastitis is not only an animal welfare problem, but also affects the yield, composition and technological properties of milk. Hence, dairy cooperatives give farmers a premium quality payment to encourage low SCC although there is no clear scientific data defining the level of SCC in bulk tank milk that is associated with additional benefits in terms of milk quality. Recent research on alternative markers for inflammatory reactions in the lactating cow, e.g. in mastitis, includes investigations of the acute phase protein, haptoglobin (Hp). So far, the content of Hp in milk has mainly been studied in relation to mastitis diagnostics, with little attention given to its importance for milk composition and technological properties. At present, Hp in milk is measured using ELISA, but this technique is not suitable for routine large-scale analysis. In recent years, optical biosensor technology has been used for automated and rapid quantitative analysis of different components in milk, but so far not for analysis of acute phase proteins. The aim of the present study was to develop a rapid and sensitive biosensor method to determine Hp in milk. An affinity sensor assay based on the interaction between Hp and haemoglobin was developed using surface plasmon resonance (SPR) biosensor technology. The assay was used to analyse Hp in composite milk samples from cows without any clinical signs of mastitis and quarter milk samples with a weak to strong reaction in the California Mastitis Test (CMT). A commercial ELISA for determination of Hp in milk was used for comparison. The limit of detection (LOD) of the biosensor assay was determined as 1.1 mg/l. Within-assay and between-day variations were determined both with bulk tank milk spiked with human Hp and with composite milk samples containing bovine Hp. Coefficients of variation varied between 3.6 and 8.6% at concentrations between 4.0 and 12 mg/l, respectively. Agreement between the results obtained by the biosensor assay and the ELISA was satisfactory; however, the results obtained by the biosensor were generally lower than the results obtained by the ELISA. Possible explanations for this observation are discussed.

Analysis of β-Lactam Antibiotics Using a Microbial Receptor Protein-based Biosensor Assay

An optical biosensor assay for detection of β-lactam antibiotics in milk based on a microbial receptor protein was developed. The assay uses a general sensor surface previously described with a small organic molecule (H1) immobilized. A conjugate between a β-lactam (cephalosporin C) and a monoclonal H1 antibody is injected across the sensor surface before injection of the sample mixed with receptor protein. Receptor inhibited by β-lactam residues in the milk sample will not bind to the sensor surface and the reduction in response is inversely related to the β-lactam concentration of the sample. The detection limit for a number of commonly used β-lactams was below or near the respective maximum residue limit and the relative standard deviation (CV) for penicillin G in milk was 6-12% in the interval 2.0-12.5 μ g kg-1. For application in the field further optimization is needed to solve problems related to non-specific binding to the sensor surface.

Comparison of biosensor, microbiological, immunochemical, and physical methods for detection of sulfamethazine residues in raw milk

A biosensor assay based on biospecific interaction analysis (BIA) was compared with already existing methods for detection of sulphamethazine (SMZ) residues in milk. Microbial inhibitor and receptor assays, an enzyme-linked immunosorbent assay (ELISA), high-pressure liquid chromatography (HPLC), and BIA were used to analyze milk samples from SMZ-treated cows. The results of the commercially available tests (Delvotest SP Special, BR-test Blue Star, Charm II test) were in agreement with the claimed sensitivity of the respective assays. The agreement between the quantitative methods (ELISA, HPLC, BIA) varied. The microbial inhibitor assays and BIA were also used to screen 330 tanker milk samples, All samples were negative in the inhibitor tests, whereas the BIA indicated the occurrence of less than 0.9 μg of SMZ per kg of milk in 5 samples and 1.5 ± 0.6 μg/kg in one sample, HPLC indicated the presence of SMZ in the latter sample, although the concentration was below the detection limit of the method. The advantages offered by the BIA: no sample preparation, high sensitivity, and rapid, fully automated analysis in real time make the technology an interesting alternative to existing screening methods within future food-quality control systems.

Haptoglobin and serum amyloid A in bulk tank milk in relation to raw milk quality

The aim of the present study was to evaluate relationships between the presence of the two major bovine acute phase proteins haptoglobin (Hp) and serum amyloid A (SAA) and raw milk quality parameters in bulk tank milk samples. Hp and SAA have been suggested as specific markers of mastitis but recently also as markers for raw milk quality. Since mastitis has detrimental effects on milk quality, it is important to investigate whether the presence of Hp or SAA indicates such changes in the composition and properties of the milk. Bulk tank milk samples (n=91) were analysed for Hp, SAA, total protein, casein, whey protein, proteolysis, fat, lactose, somatic cell count and coagulating properties. Samples with detectable levels of Hp had lower casein content, casein number and lactose content, but higher proteolysis than samples without Hp. Samples with detectable levels of SAA had lower casein number and lactose content, but higher whey protein content than samples without SAA. The presence of acute phase proteins in bulk tank milk is suggested as an indicator for unfavourable changes in the milk composition, e.g. protein quality, due to udder health disturbances, with economical implications for the dairy industry.

Factors contributing to the occurrence of antimicrobial drug residues in kenyan milk

The study investigated factors contributing to the occurrence of antimicrobial drug residues in milk within four major milk production districts in Kenya. The frequency of contamination was studied among small- and large-scale dairy producers to determine if there were differences between the two types of producers. Field samples (n = 1,600) were analyzed with the improved Dutch tube diffusion test, a microbial inhibitor test (Bacillus stearothermophilus). In total, 144 and 64 samples from small- and large-scale producers, respectively, were found to contain beta-lactam antibiotics at levels exceeding the established Codex maximum residue level for penicillin G (4 microg/kg). The difference in results between the two categories of producers was found to be significant (P < 0.001). To explain the higher frequency of antibiotic contamination of milk from small-scale producers, a questionnaire was constructed and used with 220 randomly selected smallholders in the selected districts. The results suggested (i) lack of understanding of risks related to antibiotic contamination of food, (ii) poor or no treatment records, and (iii) lack of a monitoring system as major risks for contamination. It was concluded that intensification of the education among small-scale dairy producers would greatly reduce the occurrence of antimicrobial residues in milk.

Design and Use of a General Capturing Surface in Optical Biosensor Analysis of Sulphamethazine in Milk

A new optical biosensor assay, based on a general capturing surface, for detection of the antimicrobial agent sulphamethazine (SMZ) was evaluated and compared with a previously described biosensor assay. At the general surface, the immobilisation is thought to be independent of type of analyte. Monoclonal antibodies against a small molecule (hapten H1) were immobilised and used to capture a conjugate between H1 and SMZ. Polyclonal SMZ antibodies were added to the milk sample and the amount of antibodies bound to the surface was in inverse proportion to the SMZ concentration in the milk sample. The detection limit of the new assay was 0.5 microg kg -1 and within-assay repeatability was 2.4%. This is in agreement with previous results obtained when SMZ was directly immobilised on the surface. Incurred samples from SMZ-treated cows were analysed, and non-specific binding was studied by analyses of individual cows milk. The advantages of the new assay format include analyte-independent immobilisation and reg...

A Novel Rapid Enzyme Immunoassay (Fluorophos BetaScreen) for Detection of β-Lactam Residues in Ex-Farm Raw Milk

A novel, qualitative enzyme immunoassay based on fluorescence detection for determination of beta-lactam antibiotics in raw, commingled milk (Fluorophos BetaScreen E. U. test) was evaluated. A dose-response profile for penicillin G was constructed by analysis of spiked milk samples. The limit of detection, defined as the concentration of penicillin G that resulted in 95% of the samples being evaluated as positive, was 1.8 micrograms/kg. The repeatability of the assay was very high both within and between the three participating milk quality testing laboratories. In total 5,061 randomly selected tanker milk samples were analyzed with the BetaScreen test and compared with the Delvotest SP. The agreement between the two tests was 99.7%. Probably due to a higher sensitivity to penicillin G, the BetaScreen test detected almost twice as many suspect positive tanker milk samples (0.45%) as the Delvotest SP (0.26%).

Relationship between somatic cell count, polymorphonuclear leucocyte count and quality parameters in bovine bulk tank milk

The somatic cell count (SCC) in bovine bulk tank milk is presently used as an indicator of raw milk quality, reflecting the udder health status of the herd. During mastitis, SCC increases, mostly owing to an influx of polymorphonuclear leucocytes (PMN) from blood into milk, with a concomitant change in milk composition. Bulk tank milk samples were categorized according to their SCC, as well as polymorphonuclear leucocyte count (PMNC), to study relationships between SCC, PMNC and various raw milk quality traits, i.e. contents of total protein, whey protein, casein, fat and lactose, casein number, proteolysis and rheological properties. The proportion of PMN, obtained by direct microscopy, was significantly higher in samples with high SCC compared with low SCC samples. SCC and PMNC were strongly correlated, yielding a correlation coefficient of 0.85. High SCC samples had lower lactose and casein contents, lower casein number and more proteolysis than low SCC samples. Samples with high PMNC had a lower casein number than low PMNC samples. Samples with high and low SCC or PMNC did not differ in respect to rheological properties. Our results do not indicate that PMNC is a better biomarker than SCC for raw bulk tank milk quality, as previously proposed.

Short communication: Relationships between α-lactalbumin and quality traits in bulk milk

The main objective of this study was to investigate whether the α-lactalbumin (α-LA) content of bulk milk is related with some known inflammatory markers and milk quality traits. An additional objective was to study whether combining α-LA, haptoglobin (Hp), and serum amyloid A (SAA) in an acute phase index (API) could be useful as an alternative marker for bulk milk quality. For the dairy industry, it is of great importance to receive high quality bulk milk for manufacture of liquid milk and dairy products. The somatic cell count (SCC) is currently used as an indirect marker for bulk milk quality, but because it is somewhat insensitive and unspecific, interest exists in alternative markers. Bulk milk samples were analyzed for α-LA, SCC, polymorphonuclear leukocyte count, Hp, SAA, fat, lactose, total protein and casein contents, casein number, protein composition, proteolysis, and coagulating properties. No significant differences were found in SCC, polymorphonuclear leukocyte count, Hp, or SAA between milk samples containing low, medium, or high concentrations of α-LA. Differences between α-LA groups were, however, found in some milk quality traits because high α-LA concentration was related to low concentrations of α(S1)-, α(S2)-, and β-caseins and high concentrations of lactose and β-lactoglobulin. A high API was related to low lactose content and casein number. Samples with high SCC contained less casein and had a lower casein number than milk with a low SCC, and proteolysis was significantly higher in high SCC milk than in low SCC milk. Neither α-LA nor API proved to be a better marker than SCC for the quality traits investigated, and α-LA was not considered to be a useful inflammatory marker in bulk milk.