Detection of antimicrobial substances in individual cow and quarter milk samples using Delvotest microbial inhibitor tests

The use of antibiotic therapy to treat and prevent udder infections of cows during the dry period is a key component of mastitis control in many countries. At the same time, the general public is becoming increasingly aware of potential hazards from antibiotic residues in foods. Consequently, Delvotest Cow Test (Royal Gist-brocades NV, Delft, The Netherlands), an on-farm version of Delvotest P, a microbial inhibitor test for antimicrobials, is being increasingly used by farmers to assess that milk from individual cows is fit for consignment to the bulk tank. Occasional reports of unexplained positive test results have led to suggestions of possible false-positive reactions in milk from individual cows. To investigate the potential causes of such positive test results, three separate investigations were undertaken. In a field survey of unexplained positive reports from farmers, 14 milk samples from six farms that tested positive were all found to contain antibiotic residues. In more formal investigations of individual quarter milk samples from an experimental herd, none of 134 milk samples from midlactation cows yielded positive reactions; for cows that had just calved, 16 of 144 milk samples were positive, and, of those, 13 had somatic cell counts > 4,000,000/ml. Natural inhibitors were responsible for 1 positive reaction, 8 positive reactions were related to incomplete milking, and 7 samples contained beta-lactam antibiotics. Positive reactions caused by antibiotic persisted in individual quarter samples for up to 7 d postcalving compared with 4 d for milk samples from the whole udder. Delvotest was sensitive to cephalonium, the active ingredient of Cepravin Dry Cow (Mallinckrodt Veterinary Ltd., Uxbridge, United Kingdom), which is the market-leading product in the United Kingdom. Test results yielded a partial purple color reaction in the presence of 8 micrograms/kg of cephalonium and a completely purple reaction at 16 micrograms/kg. These results confirm the validity of Delvotest when used to examine composite milk samples from individual cows supplying the United Kingdom dairy industry and suggest that, with proper attention to milk withdrawal periods and complete milking, there is no obvious risk of antibiotic contamination of milk.     

A pregnancy detection assay using milk samples: Evaluation and considerations

Two experiments were conducted to evaluate a pregnancy-detection assay based on the measurement of pregnancy-associated glycoproteins (PAG) in milk samples. In experiment 1, milk samples were collected on the day of first pregnancy check (33–52 d postinsemination; n = 119) or second check (60–74 d postinsemination; n = 60). The accuracy in identification of pregnant and nonpregnant cows was 99% at first check. Only 6% of samples were found to be within an intermediate range of PAG concentrations and classified as requiring recheck by the assay. At second check, the accuracy of the assay was 98%. Fifteen percent of these samples were classified as requiring recheck. In experiments 2a (n = 17 cows) and 2b (n = 16 cows), milk and plasma samples were collected from cows at weekly intervals beginning 2 (experiment 2a) or 4 d (experiment 2b) after insemination. The earliest time point at which pregnant cows were accurately classified as pregnant by the assay was on d 30 postinsemination. A transient decline in PAG levels into the intermediate range was observed on d 46 to 72 postinsemination. This coincides with the time of recheck in experiment 1. Results obtained with the plasma samples were essentially the same. The accuracy of pregnancy identification based on milk samples from nonpregnant and pregnant cows was 99%. Levels of PAG in milk were useful in identifying 6 incidences of embryonic mortality. No consistent relationship was noted between the timing of the decline in PAG levels and the timing of luteal regression in this small number of cows.     

Occurrence of flunixin residues in bovine milk samples from the USA

5-Hydroxy-flunixin concentrations in milk samples were quantified by two commercially available screening assays – CHARM^® and enzyme-linked immunoabsorbant assay (ELISA) – to determine whether any concentrations could be detected above the tolerance limit of 2 ng g^?1 from different regions in the United States. Milk samples came from large tanker trucks hauling milk to processing plants, and had already been screened for antibiotics. Positive results for flunixin residues based on a screening assay were confirmed by ultra-HPLC with mass spectrometric detection. Of the 500 milk samples analysed in this study, one sample was found to have a 5-hydroxy-flunixin concentration greater than the tolerance limit. The results of this study indicate that flunixin residues in milk are possible. Regulatory agencies should be aware that such residues can occur, and should consider incorporating or expanding flunixin screening tests as part of routine drug monitoring in milk. Larger studies are needed to determine the true prevalence of flunixin residues in milk from other regions in the United States as well as different countries.     

Sensitivity of the Intertest, Oxoid test, Delvotest P and disc assay to antibiotics

The sensitivity of the Oxoid, Intertest, Delvotest P and disc assay to penicillin G, tetracycline, streptomycin, erythromycin, chloramphenicol, chlortetracycline, neomycin, polymyxin B, ampicillin, novobiocin and gentamicin is presented and compared with literature values for the sensitivity of cheese starter cultures to antibiotics. Recommendations are made concerning the suitability of particular methods for detecting inhibitory levels of antibiotics in milk intended for cheese or starter manufacture.     

Analytic validation of an infrared milk urea assay and effects of sample acquisition factors on milk urea results

The objective of this study was to determine if milk samples, as they are routinely collected by Ontario Dairy Herd Improvement, would yield accurate milk urea results with an infrared assay. This investigation involved analytic validation of the infrared assay and assessment of the effect of DHI routine sample acquisition factors on milk urea results. Analytic validation of an automated milk urea assay was performed by assessing the relative accuracy and precision of milk urea results produced by the Fossomatic 4000 Milk Analyzer, an infrared method of analysis, compared with the Eurochem test, an accepted reference method. Results indicated that, when interpreted at the group level, milk urea results between the infrared method and the reference test were in good agreement. The two tests shared a similar and high level of precision. Milk urea concentrations obtained from composite (metered) milk samples, and not quarter stripping samples, were most representative of concurrent serum urea concentrations. The addition of bronopol preservative did not result in a numerically important change in milk urea concentrations. Storage of preserved metered milk samples for up to 4 d at either room temperature or by refrigeration, or for up to 3 d by freezing, did not result in changes in milk urea concentrations. We concluded that milk samples, as they are routinely collected and handled by DHI, are suitable for measurement of milk urea concentrations with the infrared method of analysis if data are interpreted at the group level.     

Occurrence of synthetic musks in human breast milk samples from 12 provinces in China

The levels of 12 synthetic musks and one musk metabolite in 24 pooled human milk samples were examined in order to assess the health risks of these contaminants to breast-feeding infants of China. The 24 pooled samples comprised of 1237 individual human milk samples collected from 12 provinces of China according to WHO guidelines. Among the 13 target analytes, OTNE ([1,2,3,4,5,6,7,8-octahydro-2,3,8,8-tetramethylnaphthalen-2yl]ethan-1-one), HHCB (1,3,4,6,7,8-hexahydro-4,6,6,7,8,8-hexamethylcyclopenta[γ]-2-benzopyran), AHTN (7-acetyl-1,1,3,4,4,6-hexamethyl-1,2,3,4-tetrahydronaphthalene), musk ketone (4-tert-butyl-2,6-dimethyl-3,5-dinitroacetophenone, MK), Musk T (1,4-dioxacyclohepta decane-5,17-dione), HHCB-lactone (1,3,4,6,7,8-hexahydro-4,6,6,7,8,8-hexamethyl cyclopenta[γ]-2-benzopyran-1-one) and musk ambrette (1-(1,1-dimethylethyl)-2-methoxy-4-methyl-3,5-dinitrobenzene, MA), were found in the milk samples analysed with mean (median) concentrations of 3.96 (3.91), 18.03 (15.10), 10.30 (9.38), 4.68 (4.45), < 3.70 (< 3.70), 10.02 (9.20) and < 5.20 (< 5.20) ng g^–1 lipid weight, respectively, whereas ADBI (4-acetyl-1,1-dimethyl-6-tert-butylindan), AHDI (6-acetyl-1,1,2,3,3,5-hexamethylindan), ATII (5-acetyl-1,1,2,6-tetramethyl-3-isopropylindan), musk xylene (1-tert-butyl-3,5-dimethyl-2,4,6-trinitrobenzene, MX), musk tibetene (1-tert-butyl-3,4,5-trimethyl-2,6-dinitrobenzene, MT) and musk moskene (1,1,3,3,5-pentamethyl-4,6-dinotroindane, MM) were not detected. Significantly positive associations were observed in concentration levels between HHCB and AHTN (p < 0.001), HHCB and HHCB-lactone (p < 0.05), AHTN and HHCB-lactone (p < 0.001), and MK and OTNE (p < 0.05). No statistical difference was found in musk concentrations between rural and urban areas in China (p > 0.05). The mean (median) estimated daily intakes by infants were 20.5 (20.2), 93.4 (78.2), 53.4 (48.6), 24.2 (23.0) and 51.9 (47.6) ng kg^–1 body weight for OTNE, HHCB, AHTN, MK and HHCB-lactone, respectively. The musk exposure levels of infants in China via breast feeding were very low according to the current toxicological information.     

Occurrence of aflatoxin M1 in randomly selected North African milk and cheese samples

Forty-nine samples of raw cow's milk and 20 samples of fresh white soft cheese were collected directly from 20 local dairy factories in the north-west of Libya and analysed for the presence of aflatoxin M₁ (AFM₁). The samples were analysed using a high-performance liquid chromatography technique for toxin detection and quantification. Thirty-five of the 49 milk samples (71.4%) showed AFM₁ levels between 0.03 and 3.13 ng ml^-1 milk. Multiple analyses of five milk samples free of AFM₁ artificially contaminated with concentrations of AFM₁ at 0.01, 0.05, 0.1, 1.0 and 3.0 ng ml^-1 showed average recoveries of 66.85, 72.41, 83.29, 97.94 and 98.25%, with coefficients of variations of 3.77, 4.11, 1.57, 1.29 and 0.54%, respectively. Fifteen of 20 white soft cheese samples (75.0%) showed the presence of AFM₁ in concentrations between 0.11 and 0.52 ng g^-1 of cheese. Multiple assays of five cheese samples free of AFM₁ spiked with different concentration of AFM₁ (0.1, 0.5, 1.0 and 3.0 ng g^-1) showed average recoveries of 63.23, 78.14, 83.29 and 88.68%, with coefficients of variation of 1.53, 9.90, 4.87 and 3.79%, respectively. The concentrations of AFM₁ were lower in the cheese products than in the raw milk samples.     

Accuracy of BRT and Delvotest microbial inhibition tests as affected by composition of ewe's milk

The presence of drug residues in ewe's milk samples can be determined by microbial assays. The main limitation of these tests is the large number of false-positive results associated with them. False-positive results can be explained by the interaction of certain substances naturally existing in ewe's milk with the growth of the microorganism used in the test. In this study, milk chemical composition (fat, protein, lactose, total solids), somatic cell counts (SCCs), free fatty acid concentrations, and lactoperoxidase system components were determined in order to investigate their influence on the rate of false-positive results for the BRT and Delvotest microbiological inhibitor tests. Milk samples were obtained after morning milking of Manchega ewes at 15, 30, 45, 60, 75, 90, 105, 120, and 135 days after parturition. The animals did not receive any kind of treatment or medicated feed throughout the experiment. The false-positive rates for BRT and Delvotest were 3.75 and 2.4%, respectively. When the logistic regression model was applied, the percentages of total solids for positive samples were significantly different from those for negative samples (16.90 versus 18.42% for BRT, 16.05 versus 18.45% for Delvotest), while the SCC logarithmic transformation was significantly higher for the positive samples than for the negative samples (5.38 versus 5.11 log units for BRT, 5.32 versus 5.11 log units for Delvotest). Moreover, Delvotest-positive samples exhibited thiocyanate concentrations higher than those of Delvotest-negative samples (8.18 mg/liter versus 6.85 mg/liter). Further analyses are needed to confirm the possible presence of antimicrobial residues in this particular type of milk sample.     

Occurrence of aflatoxin M1 in retail milk samples from Bogotá, Colombia.

A study was conducted to establish the occurrence and levels of contamination of aflatoxin M1 (AFM1) in retail milk from Bogotá, Colombia. A total of 241 samples were analysed during 2004 and 2005. Samples were cleaned up by an immunoaffinity column and AFM1 was quantified by liquid chromatography with fluorescence detection. A total of 69.2 and 79.4% of the samples analysed during 2004 and 2005, respectively, were found to contain levels of AFM1 above 10 ng l(-1). Levels of contamination ranged from 10.7 to 213.0 ng l(-1) in 2004, and from 10.6 to 288.9 ng l(-1) in 2005. Despite the high incidence of AFM1 found in the milk samples analysed, all samples complied with current local regulations, which allow AFM1 content in milk up to 400 ng l(-1). However, due to the high incidence of AFM1 in milk found in the present study, it is recommended that a permanent surveillance programme be established for milk consumed in Bogotá in order to prevent milk lots containing levels above the regulatory level entering the food chain.     

Evaluation of the Delvo-X-Press assay for detecting antibiotic residues in milk samples from individual cows.

Performance of the Delvo-X-Press beta-lactam antibiotic assay was examined using bulk-tank milk samples and milk samples from individual cows. Bulk-tank milk samples fortified with bovine lactoferrin at a concentration of 1 mg/ml or more consistently tested positive. False-positive results were also obtained from bulk-tank milk samples fortified with bovine plasma at concentrations of 20 and 40%. The assay yielded positive results for milk with antibiotic concentrations as low as 2 ppb. Individual milk samples were collected from 144 healthy lactating cows and from 34 cows with chronic Staphylococcus aureus mastitis. Specificity estimates for samples from healthy and mastitic cows were 0.88 (95% confidence interval [CI], 0.82, 0.93) and 0.94 (95% CI, 0.86, 1.00), respectively. Individual milk samples were collected from three cows with experimentally induced mastitis for 21 consecutive days. False-positive results occurred as late as 12 days postchallenge. A moderate but significant (P < 0.01) positive linear correlation (r = 0.61) was observed between test result and somatic cell count (SCC) values in milk samples with SCCs of >10(6)/ml.     

Predicting bovine milk fat composition using infrared spectroscopy based on milk samples collected in winter and summer

It has recently been shown that Fourier transform infrared spectroscopy has potential for the prediction of detailed milk fat composition, even based on a limited number of observations. Therefore, there seems to be an opportunity for improvement by means of using more observations. The objective of this study was to verify whether the use of more data would add to the accuracy of predicting milk fat composition. In addition, the effect of season on modeling was quantified because large differences in milk fat composition between winter and summer samples exist. We concluded that the use of 3,622 observations does increase predictability of milk fat composition based on infrared spectroscopy. However, for fatty acids with low concentrations, the use of many observations does not increase predictability to a level at which application of the model becomes obvious. Furthermore, the effect of season on validation r-square was limited but was occasionally large on prediction bias. For fatty acids that show large differences in level and standard deviation between winter and summer, a representative sample that includes observations collected in various seasons is critical for unbiased prediction. This research shows that all major fatty acids, combined groups of fatty acids, and the ratio of saturated to unsaturated fatty acids can be predicted accurately.     

Microbiological results from milk samples obtained premilking and postmilking for the diagnosis of bovine intramammary infections.

Bacteriological culture results were compared between 336 pairs of quarter milk samples collected premilking and postmilking. Using a positive result on either premilking or postmilking samples as the definitive diagnosis, premilking sampling sensitivity was 91% for Staphylococcus aureus, 91% for coagulase-negative staphylococci, and 97% for Streptococcus other than agalactiae. Postmilking sampling sensitivities were 81, 45, and 58%, respectively, for the same pathogens. Requiring both premilking and postmilking samples for the definitive diagnosis, specificities were 92, 86, and 95% for premilking sampling alone and 96, 98, and 99% for postmilking sampling alone. Such differences in specificity would result in major differences in predictive value of a positive culture for herds with a low prevalence. Multiple isolates were significantly more common from premilking samples.     

Routine testing of producers' milk supplies for compositional quality using randomly selected samples

In a study over a number of months, involving over 500 milk producers in three locations, and representing bulk milk collection from cans as well as mobile and stationary refrigerated bulk milk tanks, the precision of various random sampling and testing frequencies in estimating the fat, protein and lactose content of milk was evaluated. In addition, the traditional procedure of estimating the composition of milk supplies from chemically preserved composites was compared to values obtained by analysis of fresh samples. While sampling and testing every milk collection gave the most precise estimate of milk composition for each producer, it was found that for producers using refrigerated bulk milk tanks with ex-farm collection, four random samples per month gave a precision of ±4% (or ±0.14% fat approx) for monthly fat content and ±l.2% (or ±0.04% fat approx) for annual fat content. Due to lower variability the precision for both protein and lactose estimation was much higher. The sample compositing procedure, while capable of a relatively high level of precision, did not always achieve this in practice and tended to underestimate the constituents in milk, especially protein and lactose.     

Determination of calcium in milk and water samples by using catalase enzyme electrode

A biosensor based on catalase enzyme was developed for the investigation of the effect of calcium ions on the activity of the enzyme. Calcium plays an activator role for the catalase enzyme that catalyses the degradation of hydrogen peroxide to O₂ and H₂O. Determination method of the effect of calcium ion on the activity of the enzyme was based on the assay of the differences on the responses of the biosensor in the absence and the presence of calcium in the reaction medium. The biosensor had a linear relation to calcium concentrations and good measurement correlation between 1 and 10 mM with 1 min response time. Tris–HCl buffer (pH 7.0; 50 mM) and 37 °C were obtained as the optimum working conditions. In the application studies, the biosensor was used determination of calcium level of real samples such as milk, spring and mineral water.     

Discrimination between Southern Italy and foreign milk samples using spectroscopic and analytical data

Determination of the geographical origin of foodstuffs is becoming of increasing interest to consumers and producers, since it may be used as a criterion for certifying quality, authenticity and typicality.     

A study of the pH of individual milk samples

The pH of 285 milk samples was measured from early, middle and late stages of lactation. In total, 35 individual cows were used in this study.
It was found that the average pH value for all individual samples analysed was 6.63 ± 0.08. There was no significant difference ( P  > 0.05) in mean pH between early, middle and late lactation. The overall data and that for early lactation displayed normal distributions.     

Effect of freezing on bacteriologic culturing of mastitis milk samples

The objective was to determine the effect of freezing and length of freezing in a commercial freezer on the qualitative results of bacteriologic culturing of milk collected from glands of cows with clinical or subclinical intramammary infections. A total of 182 milk samples from cows with clinical mastitis and 55 milk samples of cows with subclinical mastitis were taken from four problem herds. Samples were split into four equal sub-samples. Three of these were frozen immediately at -20 degrees C and 1 was submitted fresh for bacteriologic analysis. At 4, 8, and 16 wk after collection, samples were thawed and submitted for bacteriologic culturing. Freezing and increased length of storage resulted in 1) a decrease in the number of samples that had cultures of Escherichia coli or Actinomyces pyogenes; and 2) an increase in the number of samples that had cultures of coagulase-negative staphylococci. Freezing had no effect on streptococci and Staphylococcus aureus.     

Methods of mixing donor human milk during bottling results in fat differences between samples within a pool

The influence of milk-banking processes on nutrients in donor human milk (DHM) is largely unknown. Previous studies have measured nutrients between pools of DHM, but within-pool nutrient differences (between bottles from the same pool) have yet to be elucidated. The objective of this study was to gain a better understanding of the effect of different mixing characteristics on the distribution of fat, protein, IgA, and lysozyme in bottled, raw DHM. Pools of DHM were created in a laboratory setting according to published human milk-banking guidelines and assigned to a mixing treatment (mixing during bottling method, pooling container material, and refrigerated hold time). Four mixing protocols using glass pooling containers and a 1-h refrigerated hold time were tested: control (no mixing during bottling); manual-A (Man-A, hand swirl after pouring 3 bottles); manual-B (Man-B, hand swirl after pouring every bottle); and mechanical-G (Mech-G, continuous stirring with a magnet). As secondary objectives, we compared the effect of a glass and a plastic pooling container with mechanical mixing (mechanical-P, Mech-P), and compared refrigerated delays of 1 and 24 h before bottling with manual mixing (manual-A24, Man-A24). To control for differences in nutrient content, comparisons between treatments were made using absolute percent difference from the treatment-specific mean; and comparisons within a treatment were made using the ratio of fat content in a bottle to fat content in the first bottle of the same pool. We did not observe differences in nutrient distribution between Man-A, Man-B, and Mech-G in pools held for 1 h, but all were significantly different from the control for fat. There were no differences between glass or plastic pooling containers when mechanical mixing was used. Holding a pool in the refrigerator for 24 h before bottling created significantly greater fat distribution than holding a pool for 1 h. Outcomes were the result of controlled experiments. In summary, manual and mechanical mixing of 1,700-mL DHM pools produces similar fat and protein distributions when DHM is pooled and bottled after a 1-h hold time. When DHM is held for 24 h before bottling, more research is needed to determine the duration of initial mixing needed to reduce fat variability between bottles.