Use of the Synpor membrane filter for the separation and determination of the number of somatic cells in the milk of dairy cows using the indole DNA filtration method

The somatic cells of cow's milk were separated by filtering through nitrocellulose membrane filters (Synpor), pore size 2 to 5 micron. An indole reagent was added to the membrane filters with trapped cells, the mixture was warmed in boiling water bath for 20 min., then cooled, centrifuged (3500 X g, 15 min.), and optical density was measured at 490 nm. The relation 8 micrograms = 1 million cells was used for the conversion of DNA content in sample to the counts of cells. The average variation coefficient of the determination was 4.7% and the coefficient of correlation between the indole DNA filter method and direct microscopy on membrane filters was r = 0.997. Using the indole DNA filter method, the counts of somatic cells can be estimated from milk samples kept frozen or from filters with retained cells kept at 4 degrees C, or (for a short time--3 days) at 25 degrees C. The estimation is not distorted when the milk samples are stabilized with K2Cr2O7 or a mixture of K2Cr2O7 and HgCl2, if they are kept at 4 degrees C for eight days.     

The effects of storage and method of fixation on somatic cell counts in bovine milk.

Fresh milk samples and potassium dichromate preserved milk samples were stored at both ambient, approximately 21 degree C, and refrigerator temperatures, 3-5 degree C, for varying lengths of time before somatic cell counts were performed on an electronic particle counter. Fresh milk samples stored at ambient temperatures became unacceptable for somatic cell counting by 16 hours while those stored in the refrigerator were acceptable for up to three days. Once dichromate had been added to the milk no difference in cell counts attributable to temperature of storage were detected and there was very little change with time up to 14 days. On the average the addition of the dichromate elevated the cell counts/mL. As well a method of rapid fixation of milk involving the addition of glutaraldehyde prior to counting was evaluated. In fresh milk samples the use of glutaraldehyde as a fixative required adjustment of the threshold setting on the cell counter in order to produce results comparable to those obtained from formalin fixed samples. With dichromate preserved milk samples, glutaraldehyde fixation generally elevated the cell counts but the results were variable.     

A gas-chromatographic headspace method for the determination of acetone in bovine milk, blood and urine

An automated headspace gas-chromatographic method has been developed for the determination of acetone in the milk, blood and urine of dairy cows. Five ml samples were saturated with 2 g of sodium chloride and equilibrated for 30 min at 90 degrees C in a Hewlett-Packard HP 19395 A automatic headspace sampler. The headspace volatiles were transferred without splitting to a 25 m x 0.3 mm x 0.4 microns Carbowax column in a Shimadzu GC 9A gas chromatograph, operating isothermally at 50 degrees C. The coefficients of variation for the determination of acetone were 1.5-4.4% for urine, 10.0-24.9% for milk and 2.0-19.6% for blood. The detection limits were 0.0055 mg/100 ml for milk, 0.0072 mg/100 ml for blood and 0.0080 mg/100 ml for urine. The analysis time of 5 min per sample provided an adequate rate of throughput for routine monitoring.     

Cell counts in bulked milk supplies from dairy farms of northern Nigeria

Somatic cells in 596 milk samples collected from bulked supplies of 3 northern Nigeria dairy farms were counted by an electronic method following a standard method for the preparation of the milk samples. Mean somatic cell counts per ml indicating low level of infection were 158,597, 166,742 and 155,032 for the 3 farms, without any significant differences. Mean somatic cell counts per ml indicating herd mastitis averaged 354,768 +/- 66,348 and the pathogenic organisms isolated were Escherichia coli and Staphylocucus aureus. Counts useful for future regular monitoring of somatic cells in bulked milk supplies in northern Nigeria are presented.     

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)     

Seasonal variation of bulk milk somatic cell counts in UK dairy herds: investigations of the summer rise

Individual cow somatic cell count (SCC) patterns were explored over a one year period in 33 dairy herds to investigate the reason for a summer rise in bulk milk somatic cell counts (BMSCC). Cow test day somatic cell counts were categorised according to the magnitude of change since the previous test day reading, to examine which categories were responsible for the summer increase. Multilevel models using Markov chain Monte Carlo methods were specified to estimate the number of somatic cells/ml produced by different cell count categories. Stage of lactation and parity were accounted for in the models. There was an increase in the proportion of cows that remained above 200,000 cells/ml for two consecutive recordings in summer and this group of cows were responsible for 70.8% of the increase in somatic cells/ml produced from May to September compared with October to March. There was no evidence that a greater new infection rate (somatic cell counts moving from below 100,000 cells/ml to over 200,000 cells/ml) contributed to the increased summer bulk milk somatic cell counts. There was no indication that a general small increase in all somatic cell counts played an important role in the increased summer somatic cell counts. Markov chain Monte Carlo methods provided a valuable and flexible platform for parameter estimation in reasonably complex multilevel models.     

The effect of milk cultures on the survival of salmonellae in milk

The influence was investigated of yoghurt and cream cultures on salmonella survival in milk. Salmonella-contaminated milk was blended with yoghurt culture and kept for three hours at the temperature of 43 degrees C; the mixture with cream culture was kept for 20 hours at the temperature of 22 degrees C. The samples were then stored at a room temperature and at the temperature of 4 degrees C. The two milk cultures exerted inhibitory effects on salmonellae within the range of 92.5 to 99.8%. The inhibitory effects depended on the activity of the culture (expressed by titration acidity), storage time and temperature and on the starting concentration of salmonellae.     

Role of leukotriene B4 in the pathogenesis of Klebsiella pneumoniae-induced bovine mastitis

Mastitis was induced in 4 lactating cows by inoculation of Klebsiella pneumoniae (10(7) organisms/ml) via the teat canal. Sterile isotonic saline solution (1 ml) was instilled into designated control quarters via the teat canal. Changes in milk leukotriene B4 and C4 (LTB4, LTC4) concentrations, milk somatic cell counts, and milk bovine serum albumin concentration were monitored over a 24-hour postinoculation period. Milk LTB4 concentration before inoculation in control quarters and quarters later to be infected was 376 +/- 45 and 326 +/- 56 pg/ml of milk, respectively. A significant (P less than 0.05) increase in milk LTB4 concentration in the infected quarters was first observed at postinoculation hour 6, and milk LTB4 concentration in infected quarters generally remained significantly high through postinoculation hour 14. Thereafter, milk LTB4 concentration in infected quarters was not significantly different from the concentration in control quarters. Measurable amounts of LTC4 were not detected in the milk of either control or infected quarters. Milk bovine serum albumin concentration in the infected quarters generally was high throughout the study, as were milk somatic cell counts. The results of this study suggested that LTB4 contributes to the pathogenesis of bovine mastitis.     

The associations between milk production, milk composition and Salmonella in the bulk milk supplies of dairy farms in Ontario.

The purpose of this study was to assess changes in dairy herd milk production and milk composition associated with changes in Salmonella contamination of bulk milk on dairy farms in southwestern Ontario. Twenty-three dairy farms that had submitted milk filters for culture from which Salmonella were isolated (cases) and 23 farms that submitted Salmonella-negative milk filters (controls) were included in the study. The rolling herd averages for milk and fat of case and control farms for the months of December 1985, December 1986 and April 1987 were compared and no significant differences were detected. Case and control farms were divided into three groups (A,B,C) on the basis of Salmonella culture results of milk filters submitted at various time periods throughout the study. Daily and monthly changes in milk production and composition parameters that reflected the time periods of milk filter culture were compared. The following unconditional associations between a changing Salmonella infection status on dairy farms and changes in milk production or composition variables were significant (p less than or equal to 0.05): group A: case farms had higher plate loop counts than control farms; group B: case farms had younger cows than control farms; group C: case farms had cows with longer average days in lactation than control farms. After analytical control of confounding variables, the disappearance of Salmonella from bulk milk supplies of dairy farms was associated with a decrease in percent fat and in somatic cell count.     

Somatic cells count in cow's bulk tank milk

The objective of this study was therefore to present factors affecting somatic cell counts in bovine bulk milk as a result of intramammary infections as well as non-infectious factors. The paper presents also the impact of on-farm management practices on the level of bulk milk somatic cell counts and presents quality indicators in bulk tank milk. At the farm level bulk milk bacterial infection takes place through three main sources: bacterial contamination from the external surface of the udder and teats, from the surface of the milking equipment, and from mastitis microorganisms within the udder. The threshold of 200,000 cells/ml identifies bacteriological negative quarters of the udder. The counts of mammary pathogens in bulk tank milk are relatively low, on average not exceeding 1,000 cfu/ml. Environmental pathogens predominate in bulk tank milk samples with somatic cells count <300 × 10(3) ml.     

The effects of storage temperature on goat milk somatic cell count using the DeLaval counter

This study investigated the influence of storage temperature and storage time on goat milk somatic cell counts (SCCs) determined using the DeLaval cell counter (DCC). SCCs were measured in 40 Majorera goat milk samples using the DCC device. Samples were grouped from high score (>2,750 × 10³ cells/mL) to low score (<630 × 10³ cell/mL) according to the SCC. Each milk sample was divided into four aliquots and stored at four different temperatures (4°C, 21°C, 36°C or 45°C). The SCC was recorded every hour for 12 hours. Storage of goat milk with a high SCC for 5, 5, 2 or 1 hour at 4°C, 21°C, 36°C or 45°C, respectively, decreased the SCC value compared to fresh milk. The goat milk SCC was lower after 1 hour of storage than that determined for fresh milk at any tested temperature in low-SCC samples. The data presented herein suggest that regardless of storage temperature, goat milk samples should not be stored for more than 1 hour before measurement of SCC with a DCC device.     

Absorption-spectrophotometric determination of DNA (deoxyribonucleic acid) in milk ad modum Feulgen. Accuracy of analysis, specificity, correlation to content of cells

The Feulgen reaction is examined by absorption photometric measurements at 565 nm with correction for varying background absorption at 485 nm. This correction is done to improve the accuracy of measuring.Examinations of the accuracy of analysis for the Feulgen reaction and the direct microscopic cell counts show that the former is of the same order, when the cell count is made at a working factor of 17,000.The standard deviations expressed in percentage of the reaction value are greater the lower the reaction is, whereas the absolute standard deviation is reduced.It has been demonstrated that addition of a few (< 5) µg of DNA per ml milk can give Feulgen reaction. The coefficient of correlation between log added amount of DNA and log Feulgen reaction is 0.98.The coefficient of correlation between log Feulgen reaction and log cell content depends on the accuracy at which both determinations are made. In a determination of cell content at a working factor of 550, and several determinations of the Feulgen reaction the coefficient of correlation (r) is found to be 0.98. A cell content determination at a working factor of 20,000 and a single determination of Feulgen reaction on each milk sample yields r = 0.83.An examination of foremilk samples of the same cell content reveals on an average the highest reaction in mastitis-affected quarters which have been infected within the last 4 weeks. Quarters that have been infected for more than 4 weeks, on an average show the second highest reaction, whereas quarters of physiological cell number show the lowest reaction.The DNA-content in most cases is found to be too high compared with the number of cells counted microscopically. The DNA-content in centrifugated cells corresponds to the one calculated theoretically. The surplus DNA-content can be demonstrated in the cell-free skim milk fraction, probably originating from destroyed cells.The studies performed suggest that determination of the content of 2-deoxyribose in milk by means of the Feulgen reaction is a more correct measure of the cell content in the milk than is the microscopic cell count. Studies are being continued for illustration of these conditions.     

The effect of bacterial proteases on milk proteins

The effect of proteolytic microflora on milk protein in fresh cow's milk was studied immediately after milking. The hydrolytic activity was measured by Lowry's method. When the samples were stored for 24 and 48 hours at 4 degrees C, the average value of tyrosine increased from the initial level of 0.37 mg per ml (immediately after milking) to 0.798 mg per ml (after 24 hours) and 0.811 mg per ml (after 48 hours). In milk kept at room temperature the tyrosine values were 0.865 mg per ml and 1.21 mg per ml, respectively. Higher bacterial protease activities were recorded during the first 24 hours of storage. No relationship was statistically demonstrated between tyrosine content and the number of proteolytic microorganisms in milk.     

Somatic cell counts of ewes' milk.

The somatic cell counts of ewes' milk were determined by an electronic particle counter (Coulter Counter). Of 1408 apparently normal milk samples, 98.2% had a somatic cell count lower than 1.0 x 10(6) cells/ml and 85.8% of 254 bacteriologically positive samples had a count higher than 1.0 x 10(6) cells/ml. Values exceeding 1.0 x 10(6) cells/ml are indicative of subclinical mastitis, if samples were collected from clinically healthy mammary glands.     

Serum amyloid A in the serum and milk of ewes with mastitis induced experimentally with Staphylococcus epidermidis

Mastitis was induced experimentally in ewes with Staphylococcus epidermidis, and the concentrations of serum amyloid A (SAA) in milk and serum, and the somatic cell counts and bacteria in the milk were determined for up to 10 weeks in two experiments, each examining five infected and five control ewes. The somatic cell counts peaked eight hours after infection and preceded an increase in SAA in milk. A maximum concentration of 6460 microg/ml SAA was recorded in milk from the infected sheep, compared with a mean concentration of 1.4 microg/ml in the control sheep. The mean peak concentration of SAA in serum (206.8 microg/ml) occurred earlier (one day after infection) than in milk. The serum concentration of SAA in the healthy animals ranged from 0 to 29.4 microg/ml. There was no correlation between the concentrations of SAA in serum and milk.     

Effect of intramammary devices on the outcome of induced Escherichia coli infection of bovine mammary quarters

Eighteen Holstein cows, free of intramammary infection, were fitted with smooth (n = 9) or abraded (n = 9) intramammary devices (IMD) in 2 diagonally opposed quarters within 4 weeks after calving. The 2 other quarters of each cow were used as controls. Three to 6 weeks after IMD insertion, depending on when milk somatic cell counts returned to a base-line value of less than 4 X 10(5)/ml, all cows were subjected to bacterial challenge exposure in the front or rear quarters by intracisternal injection of about 30 colony-forming units of Escherichia coli/quarter. Challenge exposure was done immediately after milking. Three weeks after the initial bacterial exposure, the other quarter pairs were similarly challenge exposed. Quarter bacteriologic status, concentration of milk somatic cells, and clinical observations (rectal temperature, milk appearance, udder palpation, and general condition of the cow) were monitored. Infection developed in 14 of 16 (88%) quarters with smooth IMD vs 16 of 16 (100%) control quarters and in 7 of 17 (41%) quarters with abraded IMD vs 17 of 17 (100%) control quarters. The difference in infection frequency between quarters with smooth IMD and quarters with abraded IMD was significant (P less than 0.05). Protection against establishment of infection was associated with somatic cell counts greater than 8.0 X 10(5)/ml in milk collected immediately after milking (7 of 12 quarters) or 4 hours later (11 of 12 quarters). In 10 quarters (59%) of cows fitted with abraded IMD, secretory abnormalities appeared before bacterial challenge inoculation. Abnormal milk or visible blood was observed over periods varying from 2 weeks after insertion through the entire lactation.     

Evaluation of a hematology analyzer with canine and feline blood

A semiautomatic electronic blood cell counter (Sysmex F-800:Toa Medical Electronics Europa Gmbh, Hamburg, Germany) was evaluated using canine and feline blood, following the International Committee for Standardization in Hematology protocol (ICSH, 1984). Precision and overall reproducibility were acceptable for all the parameters studied except for the feline platelet count, in which overlapping of erythrocyte and platelet populations prohibited determination of an accurate platelet count. Since carry-over from canine hematocrit values and platelet counts and from feline hematocrit values was unsatisfactory, the use of a blank diluent sample between different analyses was necessary. Linearity of the analyzer was acceptable in the studied range. Thirty canine and feline blood samples were analyzed using the Sysmex F-800 and a manual method. Correlations between both methods were acceptable for all the parameters, except for feline platelet count and erythrocyte indices for both species. In the storage study, red blood cell count and hemoglobin concentration were the parameters with the longest stability (72 hours at 4 degrees C and 25 degrees C) in both species. A statistically significant increase in MCV was obtained at 12 hours post-extraction in canine samples stored at 25 degrees C and at 24 hours in refrigerated samples. Feline leucocyte counts showed a downward trend at 12 hours post-extraction at both temperatures. Canine platelet count decreased significantly at 6 hours post-extraction in samples stored at 4 degrees C. During the evaluation period, Sysmex F-800 was user friendly and appeared well suited for routine canine and feline blood cell analysis.     

The effect of the lactation period on the cell content of sheep milk]

Milk samples of 201 ewes were examined in 6 week intervals during a complete lactation period. Those samples were analyzed for the presence of pathogenic bacteria and the somatic cell count was determined. Besides, the California Mastitis Test (CMT) was performed and the udder was clinically examined. The cell counts were found to depend on the lactation period. During 6 weeks following parturition the cell count was 63,000 cells/ml. This number decreased towards the 24th week of lactation to 32,000 cells/ml. At the end of lactation this value increased again to 425,000 cells/ml. The median value of ewes with normal udder health was 56,000 cells/ml milk. For samples from which pathogenic bacteria were isolated this value was 159,000 cells/ml. The most frequent pathogens isolated from the milk samples were coagulase-negative cocci (59.6% of bacteriologically positive samples), the median number being 88,000 somatic cells/ml in these sheep. Coagulase-positive cocci were isolated in 25.3% of the samples, the median value of the cell count was 295,000 cells/ml. In 12.1% of the samples streptococci were found. The median value was 167,000 cells/ml. From the remaining 3.0% of bacteriologically positive samples Pasteurellae, E. coli and Actinomycetae were isolated. The median value of the somatic cell count was 184,000 cells/ml. We consider coagulase-positive cocci therefore as the most pathogenic bacteria for the ovine udder.     

Effect of faecal extract on the growth of Salmonella enteritidis in artificially contaminated hens' eggs.

1. Salmonella enteritidis PT 4 grew in eggs stored at 25 degrees C, but not at 10 degrees C. 2. The incidence of generalised infection of the egg contents (greater than 10(6) salmonellas/ml) was greater in eggs inoculated with cells suspended in faecal extract compared to those with cells in Ringer's solution. 3. The removal of most of the iron did not decrease the growth-promoting effect of the faecal extract.     

Hypothermic storage of equine isolated hepatocytes

The aim of the study was to establish the optimal methods for hypothermic storage of equine isolated hepatocytes. Viability of equine isolated hepatocytes after hypothermic storage was dependent on the type of storage medium as well as on the cell density in the storage suspension and the preservation period. Hepatocytes stored at 4 degrees C in Hanks' Balanced Salt Solution (HBSS) and Williams' Medium E (WE) for 24 h showed very low viability, numerous cell membrane blebs, very low attachment rate (11.9 +/- 6.5% and 34.8 +/- 19.1%, respectively) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction rate (6.4 +/- 3.9% and 25.1 +/- 14.8%, respectively). In contrast, hepatocytes stored in University of Wisconsin Solution (UW) after 24 h of storage at a density of 12.5 x 10(6) cells/ml showed high viability (over 70%), typical and intact morphology, high cell attachment rates and MTT reduction. Our findings clearly demonstrate that UW is a good preservation solution for equine isolated hepatocytes. Hepatocytes harvested from slaughterhouse organs can be stored at 4 degrees C in UW at a density of 12.5 x 10(6) cells/ml for at least 24 h without significant decrease in functional integrity.