Comparison of the effectiveness of Milkofix, a preservative preparation, with traditional preservative agents in the determination of somatic cell count in milk samples using a fluoro-optic-electronic method]

Milkofix (M), a health friendly preservative substance, to be used for milk sample preservation (Trzicky, 1990), was compared with other preservatives. Untreated milk samples (N) were tested against samples treated with sodium azide (A; 0.0085 g NaN3 and 0.0630 g NaCl), bronopol (B; 0.0100 g bronopol and 0.090 g NaCl), potassium dichromate (C; 0.0330 g K2Cr2O7 and 0.0670 g KCl) and Milkofix (M; 0.1250 g). The doses of the preservatives A, B, C and M are per 25 ml milk. The somatic cell counts (SB) were determined on a FOSSOMATIC 90 apparatus (FOSS ELECTRIC, DENMARK). In the treated milk samples taken from individual cows the values of SB counts were significantly higher than in N samples if determined within eight hours after sampling (Tab. I): in A higher by 18.6%, B by 26.3%, C by 26.4% and M by 24.3% (P less than 0.05). The significantly higher values of SB counts were recorded in bulk milk samples treated with preservatives in comparison with N samples immediately after sampling: in A by 6.0%, in B, C and M by 12.9% (P less than 0.01; Tab. II). After one-day storage of N samples at a temperature of 4 degrees C these differences are insignificant (P greater than 0.05), thus the results of N, A, B, C and M samples obtained after one-day storage at 4 degrees C can be taken as actual and mutually comparable.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tests of Milkofix, a new preservative preparation for milk samples used for infrared analysis of milk components. I. Verification of its bacteriostatic and bacteriocidal effects and its interference effects]

Hygienic, ecological and health problems of sample preservation for an analysis of basic milk components make us continually to develop a safer chemical preservative substance which will preserve the original sample composition for the time required and which will not influence the analyses. Trzicky (1990) proposed Milkofix (M), a preservative substance on the basis of silver compound. The author reports on minimum risks of the use of this preparation, in comparison with traditional preservatives. Preservative efficiency of Milkofix was compared with other preservatives: K2Cr2O7 (C), NaN3 (A) and bronopol (B). The following concentrations were used: A--0.0085 g NaN3 and 0.0630 g NaCl, B--0.0050 g bronopol and 0.0500 g NaCl, C--0.0330 g K2Cr2O7 and 0.0670 g KCl in tablet, M--0.1250 g of the mixture, all amounts are per 25 ml milk. The observed antibacterial efficiency of M could be seen in a slower decrease in actual acidity, and/or in an increase in titratable acidity in M-treated samples unlike untreated ones (N). From the starting value pH 6.3 (Fig. 1), the value of N treatment dropped to 3.8 after two days, the values of M and A treatments dropped to 4.9 after nine days and to 5.7 after twelve days, respectively. As for SH, the values increased within the same interval from 6.5 (2.5 mmol/l) to 28.6 in N, and to 22.3 in M and 9.4 in A (Fig. 3). There was a similar trend when the milk samples were stored at a temperature of 4 degrees C, but the differences between the preservation methods were not so clear in comparison with storage at a temperature of 20 degrees C (Figs. 1 and 3). The standardized SH value of 9.0 (2.5 mmol) for infraanalyzer measurements was exceeded after 24 hours in N samples, after four days in M samples and after 12 days in A samples at a temperature of 20 degrees C. The observation of the growth of microorganism counts (CPM) showed that this growth was slower in M than in N, but faster in the samples of C treatment (Fig. 5). The generative time of CPM in N made 1.6 hours, in M 2.4 hours and in C 7.9 hours. The lag phase of these mixed cultures was 24 hours in M, 60 hours in C and in N treatment the lag phase was zero.(ABSTRACT TRUNCATED AT 400 WORDS)     

The effect of aging of milk samples on the accuracy of infrared analysis of milk composition]

The effect of a decrease (and/or fermentation) in the lactose content during milk storage under different conditions was investigated on the accuracy of the results obtained on a Milko-Scan apparatus to contribute to the present knowledge of this problem. The results were in agreement with some results cited in the literature. These wavelengths are used for infrared spectrophotometry on the above apparatus: for fat 3.48 microns, for proteins 6.46 microns and for lactose 9.60 microns. Bulk milk samples used for the tests were untreated or treated with potassium dichromate, bronopol, sodium azide and Milkofix at the temperatures of storage in darkness 20 degrees C and 4 degrees C. The differences against the reference values (measured on the first day) were determined and evaluated in milk composition and characteristics as arising during milk storage. These differences were used in form of either cumulative means of differences (Figs. 1 to 5) or individual differences (Fig. 8). In the first part significant correlation coefficients (P less than 0.001) were calculated for the relationship between the variations of lactose content and the fat and protein contents: r = -0.59 and/or -0.73 (Figs. 6 and 7). This suggests that the decrease in the lactose content by 0.10% recorded by the infrared analysis and caused by lactose decomposition is accompanied by a "seeming" increase in the fat and protein content by about 0.04%. In the second part the correlation coefficients for the fat and protein contents r = -0.96 and -0.96 (P less than 0.001; Figs. 9 and 10; Tab. II) were calculated on the basis of an observation of the lactose decrease in an untreated milk sample (20 degrees C for 28 hours). These coefficients are somewhat different from the preceding ones; this is due to the lower homogeneity of the first set where the milk samples were treated in a different way, but the coefficients confirm the same conclusions. The values of the correlation coefficients for the dependence between the development of the acquired titratable acidity (SH) and the variations of fat (F), protein (P) and lactose (L) contents were as follows: r = 0.95; 0.95; -0.99 (P less than 0.001; Figs. 12, 13; Tab. II). Thus the above-mentioned "seeming" increase in the F and P contents can be explained to the extent of 92.2% from the decrease in the L content, which also causes the increase in titratable acidity to the extent of 98.0%.(ABSTRACT TRUNCATED AT 400 WORDS)     

New findings on the biotyping of Staphylococcus aureus isolated during milk processing]

A detailed analysis of biotypes of Staphylococcus aureus, as related to their origin and enterotoxigenicity, was performed, using 432 strains isolated from bulk milk, milking machines, quarter milk samples collected from mastitic cows, and cowherds and milkers. All strains coagulated rabbit blood plasma and produced thermonuclease (Tab. I). Human strains differed from bovine ones mostly in the production of alpha-haemolysin (94%) and fibrinolysin (66%). Biotypes C1 (35%) and C2 (38%) dominated clearly among the strains isolated from quarter milk samples. The findings of 13% of biotype A and 8% of biotype D suggest that other sources of udder infections than mastitic cows were involved. Almost 19% of human strains and two strains isolated from quarter milk samples were identified as the recently defined type G. The production of enterotoxins (Tab. III) of was associated mostly with strains of human origin (69%) and with biotypes G (35%) and A (31%). Three enterotoxigenic strains belonged to the biotype B and one strain was not classifiable.     

Evaluation of flow injection analysis for determination of urea in sheep's and cow's milk

Difficulties in measuring the urea content in sheep's milk often occur with spectral photometry due to the high protein and fat concentrations of the milk. In this study an enzymatic flow procedure (QuickChem 8000 Ion Analyser, Lachat Instruments, Milwaukee, USA) to determine the urea content in ovine and bovine milk was evaluated. Urea content is determined by the Berthelot reaction after splitting it enzymatically with urease. The free ammonia diffuses through a teflon membrane into a stream of reagent solutions. Detection takes place by means of a reaction between the ammonium ions with hypochlorite and salicylate producing a green colour, which is measured spectrometrically in a flow meter at 660 nm. By using a diffusion cell chemical deproteinisation of milk is not necessary and capacity is high. The assessed procedure exhibited high accuracy and precision and reached a sample capacity of 55 samples an hour. Storage of the milk samples for several days as well as chemical preservation with bronopol had no effect on the measurement procedure. Due to the complexity of the apparatus and the costs associated therewith, the device proves less suitable for routine diagnostics but rather serves as a reference method for the measurement of urea concentration in milk.     

The effect of cellulase on calves fed an acidified milk diet]

The purpose of this research was to establish the influence of 3 j Cx cellulase applied per gram of COT concentrate mixture, fed in combination with a milk diet acidified by formic acid to the value of pH 4.6, on calf growth performance in one metabolism and two field experiments. In the metabolism experiment two groups of calves, with six animals in each, were fed acidified whole milk, which was diluted stage by stage till weaning at 60 days of age. The average live weight gain in the control at the end of the milk feeding period, i.e. from 14 to 60 days of age, was 29.90 kg. This corresponds to a daily live weight gain of 0.650 g. The total live weight gain of male calves in the experimental group was 29.30 kg, corresponding to a daily live weight gain of 0.638 g (Tab. I). Tab. II shows the average feed and nutrient intakes per kg live weight gain. The calves which received the enzyme supplement tend to have the higher feed conversion rate. During the forage feeding period, i.e. from 61 to 90 days of age, the average daily live weight gains were 1.10 kg and 0.980 kg in the control and experimental groups, respectively (Tab. III). The average live weight of 90 days old male calves was 107.70 kg and 103.90 kg in the control and experimental groups, respectively. The amount of consumed nutrients (digestible protein and starch units); in relation to the total feed intakes, is lower in the experimental groups, which proves the higher feed conversion rate (P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

Relation of lactose levels in milk and indicators of mammary gland health in the first third of lactation]

A total of 662 bucket milk samples from cows of two breed groups were examined: red and white breed = Bohemian Pied cattle with different genetic proportions of Ayrshire and Red Holstein improvement breeds; black and white breed = Black and White Lowland breed and different degree of absorptive crossing with a genetic proportion of the Holstein breed. Samples of daily milk yields were taken in the first three months of lactation once a month within a year. A possibility of using lactose content as an auxiliary indicator for detection of the mammary gland secretion disorders in the initial lactation stage was evaluated. The average values of the different indicators and their variability are summarized in Tab. I showing also the significance in a statistical model of included effects. Lactose content (L) was 4.88 +/- 0.20%, chloride content (Cl-) 113.7 +/- 22.4 mg/100 ml, somatic cell (SC) count 474 +/- 805 thousand/ml, SC count log corresponds to the geometrical mean of 234 thousand/ml, titratable acidity (SH) 7.34 +/- 0.83 x 2.5 mmol/l, chloride-lactose ratio (ClL) 2.27 +/- 0.51, conductivity (gamma) 442.4 +/- 34.5 mS/m and mastitis test (MT-NK) 0.72 +/- 1.18. The efficiency of the used statistical model was highest for Cl- content (Tab. I, R2 = 0.41), and it was lowest for SC counts (R2 = 0.07), while it increased to the twofold value (R2 = 0.15) after logarithmic transformation of SC counts. The breed group exerted a significant effect on Cl-, SC, log SC, SH, ClL, gamma and MT-NK (Tab. I). The breed group of red and white cows (Tab. II) had higher component contents and better indicators of the udder health state (Cl-, SC, log SC, ClL, gamma and MT-NK). The month of lactation influenced significantly SC, log SC, SH and gamma (Tab. I). A decrease in SC counts with the accruing month of lactation was observed (Tab. II), the trend of gamma and SH was opposite. The effect of lactation number was found to be significant for L, SC, log SC, SH, ClL, gamma and MT-NK (Tab. I). A tendency of a gradual decrease with the lactation number was observed in these indicators: L, SH, proteins and solids-non-fat (Fig. 1), while Cl-, gamma and ClL showed an opposite tendency. The year season influenced significantly L, log SC, SH, ClL, gamma and MT-NK (Tab. I, Fig. 2).(ABSTRACT TRUNCATED AT 400 WORDS)     

Flow injection analysis for the determination of urea in cow's milk

An inexpensive and easily automated flow injection method for determination of urea in cow’s milk was evaluated. Urea is hydrolysed by urease and in a gas diffusion cell the ammonia formed passes a membrane into an indicator solution. The resulting colour change of the indicator is measured at 590 nm.The repeatability of the analysis, expressed as the coefficient of variation (C.V.), was between 0.5 and 1.2%. Measured (y) and expected (x) milk urea concentrations after addition of known amounts of urea were related according to the equation y = 1.00× – 0.12 with a C.V. for the regression of 1.8%. Recommended amounts (0.02 %) of the preservative bronopol (2-bromo-2-nitropropane-1,3-diol) added to the milk did not affect the results (P > 0.05).     

Detection by immunomagnetic PCR of Mycobacterium avium subsp. paratuberculosis in milk from dairy goats in Norway

Milk samples from 340 individual goats in 34 dairy herds throughout Norway were examined for Mycobacterium avium subsp. paratuberculosis (M.a. paratuberculosis) by culture and immunomagnetic separation combined with PCR (IMS-PCR). The samples included three categories; (A) vaccinated dairy goats in herds with paratuberculosis; (B) vaccinated dairy goats in herds with no history of paratuberculosis; (C) unvaccinated goats in herds with no history of paratuberculosis.Viable M.a. paratuberculosis were not detected by culture in any sample, but 24 samples (7.1%) tested positive by IMS-PCR when the PCR products were visualised by dot blot hybridisation. PCR products from five milk samples originating from five different herds were sequenced; all showed 99% homology with the IS900 sequence from M.a. paratuberculosis.M.a. paratuberculosis were detected in all sampled categories. The percentage of IMS-PCR positive samples from herds where paratuberculosis had previously been reported was significantly lower than from herds where the infection had never been diagnosed (3.3 and 9.1%, respectively, P=0.048). Similar proportions of milk samples from vaccinated and non-vaccinated goats tested positive for the presence of M.a. paratuberculosis. Vaccinated goats older than 4 years tested positive more often than vaccinated animals less than 2 years old. Samples collected in May tested significantly more often positive than milk sampled during February-March (13.8 and 2.9%, respectively, P=0.001). This study showed that raw goats' milk in Norway might be contaminated with M.a. paratuberculosis.     

Isobutylidene diurea, a new NPN source for ruminants. 3. Excretion of isobutylidene diurea following feeding of N-15 isobutylidene diurea to lactating cows

2 experimental cows received isobutylidenedi urea added to a natural diet in amounts of 175 g (I) and 730 g (II) per day for a period of several weeks before the trial was started. On the 1st day of experiment the morning dose was labelled with 5.05 g of excess 15N. 8 hrs after the beginning of the trial of 15N level in the TCE soluble portion of blood plasma (TCE=trichloroacetic acid) increased and remained at an elevated level until the 36th hour of experiment. Similarly, the values for maximum urinary 15N concentrations were maintained for a prolonged period of time. Isobutylidenedi urea was excreted with the urine in rates related to its solubility. Only small percentages of the 15N intake were excreted in the TCE soluble portion of the milk (cow I: 0.03%; cow II: 0.05%). The 15N-labelling of milk protein provides evidence for the fact that nitrogen from IBDU is utilized for the synthesis of milk in the cows. The amount of urea in milk averaged 400 mg per litre. None of the milk samples tested contained IBDU.     

Use of polychlorinated biphenyl congener analysis for monitoring food and raw material of animal origin]

Occurrence of individual polychlorinated biphenyl (PCB) congeners in the environment, foodstuffs and other biological materials was assessed. Analysis of specific PCB congeners (28, 52, 101, 138, 153, 180), occurring in animal raw materials and foodstuffs most frequently, has been implemented. Sample processing, isolation of fat from milk, meat, organs, fat tissue and eggs, and separation of PCB from fats using the sorbents Florisil or Ekosorb (a new Czechoslovak sorbent based on modified silica gel, Kavalier Glassworks, Votice) are described in detail. Individual PCB congeners were determined by capillary gas chromatography (gas chromatograph Varian VISTA 6,000, equipped with a 63Ni ECD; silica capillary column SPB-5, 30 m x 0.32 mm I.D., 0.25 micron film; column temperature programme: 60 degrees C for 2 min, then to 250 degrees C at 20 degrees C/min and held for 13 min; splitless injection). Chromatograms of commercial chemicals Delor 103 and Delor 106 (corresponding to Aroclor 1242 and 1260, respectively), of a mixture of six specific PCB congener standards and of a PCB--containing milk sample are presented in Fig. 1-3. Methods of PCBs estimation, currently used in Czechoslovakia, and benefits of congeneric analysis of PCBs (reproducibility of results, quantification of individual congeners) are discussed. Analysis of specific PCB congeners is used for the assay of PCBs in foodstuffs and investigations of PCBs dynamics in food chains and distribution and accumulation of PCBs in animal organisms. Contents of specific PCB congeners in milk, pork and pig liver and kidney samples are given in Tab. II.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evaluation of a Rapid Method for the Determination of Urea in Cow’S Milk

A rapid method for the determination of urea in milk was evaluated. The method employed was a urease/glutamate dehydrogenase procedure where the decrease in absorbante of NADH is monitored spectrophotometrically at 340 nm. The repeatability expressed as a coefficient of variation, was 1.4 % and 0.8 % for whole milk and skim milk, respectively. The accuracy of the method for urea determinations in whole milk was satisfactory (coefficient of variation, 2.6 %). Adding the preservative bronopol (0.02%) to the milk or freezing of the milk samples for one week did not alter urea levels significantly (P > 0.05). The storage of preserved whole milk in a refrigerator for 14 days, however, increased milk urea levels measured by 3.1 % (P < 0.001).It was concluded that the method evaluated is simple, rapid (40–50 samples/h) and yields reliable results also when whole milk is used. Since the determination of milk urea may be useful in assessing the feeding of energy and protein, the method is suitable for integration in the existing milk recording system in Sweden.     

Body weight and selected hematologic and biochemical parameters in calves fed various diets during milk feeding]

The objective of the present paper was to obtain haematological and biochemical parameters of calves during their milk feeding. Another goal was to compare a current feed ration containing the commercial feed mixture Biosan with two alternative rations comprising skimmed milk and fat supplement. The effect of milk drink acidification and the effect of the physical form of fat added to skimmed milk were investigated. the calves were divided into six groups, by 6 to 8 calves each (P1-Biosan, PP1-acid variant, P2-skimmed milk+fat concentrate powder KMKS, PP2-acid variant, P3-skimmed milk+fat paste, PP3-acid variant). The highest average daily liveweight gains were recorded in calves fed on unacidified skimmed milk with fat paste supplement (1.074 kg)-Tab. I. The highest average daily liveweight gains were observed in calves fed on fat paste enriched milk drink (P3) while the calves administered milk with fat concentrate powder (P2) had only slightly higher average daily weight gains than the calves on Biosan diet (group P1). The growth of daily weight gains as a result of diets containing acidified milk (PP1 and PP2) is also a positive finding; this growth is even statistically significant in the former case (P < 0.05). The calves showed hypohaemoglobinaemia from the beginning of the experiment (51.3-69.0 g/l) and the low haematocrit value (0.29-0.43 l/l), Tab. II, while the plasma iron level was paradoxically high (Tab. V).(ABSTRACT TRUNCATED AT 250 WORDS)     

Relation between milk production and biochemical indicators in high- and low-producing cows]

23 cows of the Holstein-Friesian breed during second lactation were used in the experiment. All animals were housed in one stable with tying. The blood was collected from each cow four times during first to third, fourth to sixth, seventh to ninth and above nine months of lactation. After 305 days of lactation, the milk cows were classified as with high (n = 14) and lower performance (n = 9), the limit value was 5000 kg of milk. The difference in milk production between groups was highly significant even during the period of the first 90 days (501 kg) and this difference increased for 305 days of lactation to 1538 kg (Tab. I); the dairy cows with high performance produced 6162 kg of milk per 305 days and that with the lower performance gave 4624 kg during the same period. Another significant differences occurred in the fat contents per kg and in the amount of milk converted on 4% fat content. During the first stage of lactation a slightly higher haemoglobine level was found in cows with high performance, the opposing results were obtained in another studies (Tab. II), the highest difference was recorded in the course of 7th to 9th month. The level of total protein was, except 7th to 9th month, always higher in cows of the first group. The difference was significant at the beginning of lactation. Insulin concentration (INS) exhibited identical tendency for the whole lactation--dairy cows with high milk production exhibited lower values, significant differences were recorded in the first observation at the beginning of lactation and in the period above 9 months. Similarly as in case of insulin, it is also in hormones of thyroid gland, the cows with high milk capacity had the lower values (Tab. III). The highest differences in triiodothyronine (T3) concentration were recorded in the second half of lactation, significance ranged from 7th to 9th month of lactation. In thyroxine content (T4), marked differences were recorded during the first and last observations, the difference was highly statistically significant during the seventh to ninth month of lactation. The levels of cyclic adenosine monophosphate (AMP) were higher for the whole experiment in animals with lower milk production. Tab. IV gives the correlation coefficient of different parameters between periods under study. Almost all parameters are in a close positive relation, the closest dependences were observed between second, third and fourth periods in hormones of thyreoidea. The most significances were recorded in adenosine monophosphate, thus confirming its stability of minimum variance between observations.(ABSTRACT TRUNCATED AT 400 WORDS)     

A new dried milk sampling technique and its application for progesterone detection in cows

A new method for milk sample collection and storage, based on a dried milk sampling technique, is proposed. The method includes application of a whole milk sample to a porous membrane followed by drying. One hundred whole milk samples (dried and liquid) taken on day 21 post insemination were analysed for progesterone by ELISA and results for both dried and liquid samples were well correlated (r = 0.911). Milk progesterone ELISA accuracy for pregnancy diagnosis in cows was 87%.     

Elimination kinetics of chlorhexidine in milk following intramammary infusion to stop lactation in mastitic mammary gland quarters of cows

OBJECTIVE: To evaluate the elimination kinetics of chlorhexidine in milk when used as an intramammary infusion to stop lactation in cows. DESIGN: Prospective study. ANIMALS: 6 cows. PROCEDURE: The study was performed in 2 phases. Three cows were studied in each phase. All cows were treated with chlorhexidine suspension by infusion into a mastitic mammary gland quarter after 2 milkings 24 hours apart. Foremilk samples (100 mL) were collected from treated and untreated (controls) mammary gland quarters of each cow. Chlorhexidine was extracted from raw milk, and residue concentrations were quantified by use of high-performance liquid chromatography. Foremilk samples from days 2, 5, and 8 were analyzed in phase I, and samples from time 0 and days 3, 7, 14, 21, 28, 35, and 42 were analyzed in phase II. RESULTS: In phases I and II, there was no quantifiable transference of chlorhexidine to milk in untreated mammary gland quarters. Measurable chlorhexidine residues were found in milk from treated mammary gland quarters of 2 cows throughout the 42-day sample period in phase II. Estimated mean elimination half-life for chlorhexidine in milk was 11.5 days. CONCLUSIONS AND CLINICAL RELEVANCE: On the basis of the long elimination half-life of chlorhexidine in milk from treated mammary gland quarters, the lack of human dietary exposure data to suggest a food tolerance for chlorhexidine in food products, and the Food and Drug Administration's published zero tolerance for chlorhexidine in uncooked edible calf tissues, we do not recommend extralabel use of chlorhexidine suspension as a treatment to stop lactation in mastitic mammary gland quarters of cows.     

Improved detection of Mycobacterium avium subsp. paratuberculosis In milk by immunomagnetic PCR

The potential use of a novel immunomagnetic PCR (IMS-PCR) technique as a rapid method to screen milk samples for the presence of Mycobacterium avium subsp. paratuberculosis (M. ptb) was assessed. Immunomagnetic separation (IMS) for M. ptb, developed at Queen's University, Belfast, was applied to milk samples prior to IS900 PCR in order to selectively concentrate any M. ptb cells present and, at the same time, separate the cells from constituents of milk likely to inhibit subsequent PCR. This increased the sensitivity of IS900 PCR. IMS-PCR sensitivity could be further increased by initial centrifugation (2500 g for 20 min) of larger volumes of milk (10 and 50 ml), and resuspension of the sediment into a 1 ml volume appropriate for IMS treatment. Following IMS, template DNA for IS900 PCR was obtained by heating the bead-cell suspension in a thermal cycler at 100 degrees C for 15 min. It was estimated that the IMS-PCR assay could detect approximately 10(3)CFU of M. ptb per 50 ml of milk (equivalent to 20 CFU/ml), whereas the minimum detection limit of direct IS900 PCR was estimated at 10(5)CFU of M. ptb per 50 ml (equivalent to 2000 CFU/ml). A blind trial was carried out in which a total of 40 spiked (10(6)CFU M. ptb) and unspiked, raw and laboratory-pasteurised milk samples were independently tested by IMS-PCR and conventional IS900 PCR. IMS-PCR correctly identified 97. 5% of milk samples (sensitivity 100%, specificity 95%), including spiked milk samples before and after laboratory-pasteurisation. One false positive result was obtained which may have resulted from carryover between samples during the IMS procedure. Conventional IS900 PCR correctly identified only 72.5% of the same 40 milk samples (sensitivity 23%, specificity 100%). IMS-PCR was also shown to be capable of detecting natural M. ptb infection in raw sheep's milk, and raw and commercially pasteurised cows' milk.     

Development of a real-time PCR for detection of Mycoplasma bovis in bovine milk and lung samples.

A real-time polymerase chain reaction (PCR) assay using hybridization probes on a LightCycler platform was developed for detection of Mycoplasma bovis from individual bovine mastitis milk and pneumonic lung tissues. The detection limit was 550 colony forming units (cfu)/ml of milk and 650 cfu/25 mg of lung tissue. A panel of bovine Mycoplasma and of other bovine-origin bacteria were tested; only M. bovis strains were positive, with a melting peak of 66.6 degrees C. Mycoplasma agalactiae PG2 was also positive and could be distinguished because it had a melting peak of 63.1 degrees C. In validation testing of clinical samples, the relative sensitivity and specificity were 100% and 99.3% for individual milks and 96.6% and 100% for the lung tissue. Using M. bovis real-time PCR, the M. bovis culture-positive milk samples were estimated to contain between 5 x 10(4) and 7.7 x 10(8) cfu/ml and the M. bovis culture-positive lungs between 1 x 10(3) and 1 x 10(9) cfu/25 mg. Isolation, confirmed with the real-time PCR and colony fluorescent antibody test, showed that at the herd level, the proportion of samples positive for M. bovis isolation in mastitis milk samples submitted to the Mastitis Laboratory, Animal Health Laboratory, University of Guelph, Ontario, Canada, was 2.4% (5/201). We conclude that this probe-based real-time PCR assay is a sensitive, specific, and rapid method to identify M. bovis infection in bovine milk and pneumonic lungs.     

The ovarian response and fertility in cows with acidosis induced by acetic acid]

The effects of acetic acid administered at an amount of 300 to 600 g (5 to 10 mol) to the rumen of breeding cows, were investigated on ovulation, conception and progesterone levels in the blood and milk of cows with cloprostenol-induced (Oestrophan Spofa) oestrus at a dose of 500 micrograms i.m. In the group of 15 cows exposed to the acetic acid load five cows got in calf after the first insemination (33.3%), and 12 cows (80.0%) after all inseminations in 37.6 days after cloprostenol administration, with the insemination index 1.67 (Tab. I). In the control group (five cows) four cows (80.0%) got in calf after the first insemination, in total all five breeding cows got in calf in 20.6 days after cloprostenol administration, with the insemination index 1.2. In the experimental group of 15 cows a clinical examination of ovaries on day 7 after insemination revealed ovulation disorders in eight cows, that means in 53.3% of the animals (Tab. II). No ovulation disorders were observed in the control group of five cows. Progesterone levels in the blood showed high variability (Tab. III). In the group of cows administered acetic acid they were by more than a half lower (1.49; 0.67; 1.53 per ml) on days 7, 14 and 21 after insemination in comparison with the control group (3.35; 2.5; 3.38 ng per ml). The average progesterone levels in milk (Tab. IV) were 1.27 and 1.53 on day 7, 6.74 and 7.27 on day 14 and 3.52 and 11.85 ng per ml on day 21, respectively, the higher values apply to the control. It was not possible to evaluate reliably from the progesterone levels in the blood and milk if ovulation took place and if the corpus luteum was developing (Tab. V and VI). The clinical control of ovaries on days 7 and 8 after oestrus and insemination was more reliable to determine the ovulation disorders than the progesterone determination in the blood and milk of cows.     

The significance of mycotoxin assimilation for the contamination of milk and milk products

The two possible pathways contaminating milk and milk products with mycotoxins are either the secretory or post-secretory route. The latter is of only little importance due to cooling conditions in production and storage. A secretory contamination can only occur with such mycotoxins, which undergo no complete degradation through their passage into the milk. From the mycotoxins, present in cow's feed; virtually only aflatoxin B1 yields a milkborne metabolite, the aflatoxin M1. The carry over rate is low (2 +/- 1%), but can be enhanced by polyhalogenated biphenyls, also present in the forage. Under normal conditions, however, this enhancement will not be measurable due to low equimolar concentrations of both reactants. The aflatoxin M1 content in herd's bulk milk depends exclusively on the content of the precursor aflatoxin B1 in the ration of the cow and is with less than 10 ng/kg fairly low at present in the Federal Republic of Germany. A careful supervision of the imported feed ingredients for mixed feed, however, will ensure to keep those batches out of dairy cow feeding which exceed a certain level of aflatoxin. The legal threshold is 10 micrograms/kg, being even too high to ensure a milk containing less than 10 ng/kg under high energy feeding conditions. The discussed thresholds for aflatoxin M1 in milk are 50 and 10 ng/kg resp., the latter value is scheduled for milk used in infant nutrition. To keep this low concentration the intake of aflatoxin B1 must be less than 2 micrograms/kg of the daily ration.