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.

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.

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 analysis of beta-lactams in milk using the carboxypeptidase activity of a bacterial penicillin binding protein.

The applicability of a beta-lactam receptor protein for detection of beta-lactam antibiotics in milk using surface plasmon resonance (SPR) biosensor technology was investigated. The advantage of using a receptor protein instead of antibodies for detection of beta-lactams is that a generic assay, specific for the active form of the beta-lactam structure, is obtained. Two assays based on the enzymatic activity of the DD-carboxypeptidase from Actinomadura R39 were developed, using a Biacore SPR biosensor. The carboxypeptidase converts a tri-peptide into a di-peptide, a reaction which is inhibited in the presence of beta-lactams. Polyclonal antibodies against the 2 peptides were developed and used to measure the amount of enzymatic product formed (di-peptide assay) or the amount of remaining enzymatic substrate (tri-peptide assay), respectively. The 2 assays showed similar performances with respect to detection limits (1.2 and 1.5 microg/kg, respectively) and precision (coefficient of variation <5%) for penicillin G in milk. Several other beta-lactams were detected at or near their respective maximum residue limit. Furthermore, the 2 peptide assays were evaluated against 5 commercial kit tests in the screening of 195 producer milk samples. The biosensor assays showed 0% false-negative and 27% false-positive results, whereas the figures were 0% false-negative and 27-53% false-positive results for other screening tests investigated.

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.

Affinity and rate constants for interactions of bovine folate-binding protein and folate derivatives determined by optical biosensor technology. Effect of stereoselectivity.

The interactions between bovine folate-binding protein (FBP) and different folate derivatives in pure diastereoisomeric forms were studied at pH 7.4 by a surface plasmon resonance technology (Biacore). The results show that folic acid had the most rapid association rate (k(a) = 1.0 x 10(6) M(-)(1) s(-)(1)), whereas (6S)-5-HCO-5,6,7,8-tetrahydrofolic acid had the most rapid dissociation rate (k(d) = 3.2 x l0(-)(3) s(-)(1)). The equilibrium dissociation constant (K(D)), calculated from the quotient of k(d)/k(a), showed that the two forms of folates not occurring in nature, that is, folic acid and (6R)-5-CH(3)-5,6,7,8-tetrahydrofolic acid, had the highest affinities for FBP, 20 and 160 pmol/L, respectively. The results thus show that there were great differences in the interactions between folate-binding protein and the major forms of folate derivatives. The nutritional implications of these differences are discussed.

Biosensor analysis of beta-lactams in milk: comparison with microbiological, immunological, and receptor-based screening methods.

Two recently developed surface plasmon resonance biosensor assays for detection of beta-lactams in milk were used to screen raw producer milk samples. Both assays use a beta-lactam receptor protein with carboxypeptidase activity for detection. The results of the biosensor assays were compared with those of various commercial screening tests, i.e., the Delvotest SP, Penzym S, Beta-STAR, SNAP, and Parallux. The results of the 2 biosensor assays showed good agreement with those of the other screening tests. Of 195 analyzed milk samples, the results of only 5 samples differed between the assays. Additionally, 30 milk samples with both negative and positive results in the screening assays were analyzed by liquid chromatography for identification and quantification of any beta-lactam residues. All screening tests showed 0% false-negative results with 15 incurred samples containing between 4.0 and 268 microg/kg penicillin G. The biosensor assays showed 27% positive results (false violatives) with 15 producer milk samples containing penicillin G concentrations between 0 and 3.6 microg/kg, i.e., below maximum residue limit. This figure varied between 27 and 53% for the other screening tests.

Determination of beta-lactams in milk using a surface plasmon resonance-based biosensor.

Two surface plasmon resonance (SPR)-based biosensor assays for detection of beta-lactam antibiotics in milk are reported. The assays are based on the enzymatic activity of a carboxypeptidase converting a 3-peptide into a 2-peptide, a reaction that is inhibited in the presence of beta-lactams. Antibodies were used to measure either the amount of formed enzymatic product or the amount of remaining enzymatic substrate. Both assays detected different beta-lactams at or below European maximum residue limits (MRLs), and the detection limit for penicillin G was 1.2 microg/kg and 1.5 microg/kg for the 2- and 3-peptide assays, respectively. The precision (CV) was < 5%, both within and between assays at the penicillin G MRL (4 microg/kg). The biosensor results obtained upon analysis of incurred milk samples were compared with results obtained by liquid chromatography (HPLC), and the method agreements were, in general, good.

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.

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

A novel, qualitative enzyme immunoassay based on fluorescence detection for determination of (ß-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 µg/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%).

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.