Determination of the number of somatic cells in milk using the rapid diphenylamine DNA filter method

The rapid reaction of the diphenylamine agent with DNA was used for the determination of the counts of somatic cells in cow's milk, using the DNA filter method. The method is based on the filtration of a warmed (65-70 degrees C) mixture of milk with Triton X-100 through the Synpor nitrocellulose membrane filter, pore size 2 to 5 microns, and subsequent DNA determination of the collected somatic cells by the colour reaction of diphenylamine. A 2ml quantity of distilled water and 4 ml of diphenylamine reagent were added to the membrane filters with somatic cells. The mixture is warmed in water bath at 90 to 100 degrees C for 20 min., then it is cooled, centrifuged (3500 X g, 15 min.), and the optical density is measured at 595 nm. The relation 8 micrograms = 1 million cells was used for the conversion of DNA content to the counts of cells. The average variation coefficient of the determination was 5.9% and the coefficient of correlation between the diphenylamine DNA filter method and the direct microscopy of the somatic cells on membrane filters was r = 0.997. Using the diphenylamine DNA filter method, the counts of somatic cells can also be determined from milk samples stored in frozen condition or from the filters with collected cells kept at the temperature of 4 degrees C (10 days) or 25 degrees C (3 days). Milk stabilized with formaldehyde can also be used for the determination if stored at 4 degrees C.     

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)     

Reduction of aflatoxin M1 in milk using hydrogen peroxide and hydrogen peroxide plus heat treatment.

Use of hydrogen peroxide (H2O2) under various conditions of temperature and time to inactivate aflatoxin M1 in artificially contaminated raw milk was examined. The degree of inactivation was measured by Enzym Linked Immunosorbent Assay (ELISA). It appeared that there is no change in the content of aflatoxin M1 in milk contained H2O2 and examined after 24 hours without heat treatment and in contaminated milk without add of H2O2 and heat treated treatment at (63 degrees C for 30 min, 75 degrees C for 15 sec) but slight inactivation (4.3%) was obtained in milk boiled for 5 min without H2O2. Maximum inactivation (27.8%, 28.8% and 45.1%) were obtained using 1% H2O2 followed by heat treatment at 36 degrees C for 30 min, 75 degrees C for 15 sec and boiling for 5 min respectively.     

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.     

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.     

Bactericidal effects of ozone at nonspermicidal concentrations.

A study was conducted to assess the use of ozone (O3) to control pathogens or contaminants of concern to animal breeders and regulatory officials. In separate experiments, samples of fresh bovine semen and Pseudomonas aeruginosa, Escherichia coli, or Campylobacter fetus subsp. venerealis were diluted with antibiotic-free milk (10(6) sperm and 10(6) organisms/mL of diluted semen), exposed in the previous day to a constantly monitored level of 5, 10, 15, or 20 micrograms/mL of O3 for 3-5 min. After 10 min at 30 degrees C, sperm motility was assessed and the samples cooled to 5 degrees C. Two and 18 h after the beginning of cooling, aliquots of each semen sample were evaluated for motility and cultured for organisms. Reductions were observed in P. aeruginosa and E. coli colony counts of 2 logs, and in C. fetus of 5 logs, after exposure for 2 h to O3 at a concentration of 5 micrograms/mL that had a moderate effect on sperm motility (reduction of 20%). Fewer than 100 colonies, i.e., a 4 logs reduction of all bacteria, were counted after dilution with ozonized-treated milk at 20 micrograms/mL of O3. However, this concentration of O3 reduced sperm motility by 50% 10 min after dilution. The results of these experiments indicate that a concentration and exposure time to O3 can be selected to reduce P. aeruginosa, E. coli, and C. fetus in contaminated bull semen diluted with milk while having only minimal effects on sperm motility.     

Comparisons of antibiotic combinations to control Pseudomonas aeruginosa in bovine semen.

Raw semen experimentally contaminated with 10(6) Pseudomonas aeruginosa cells per milliliter was processed for use in artificial insemination (AI) using three different antibiotic combinations: a) gentamicin, lincomycin, spectinomycin and tylosin (GLST) directly added to contaminated raw semen followed by dilution with whole milk or egg yolk Tris containing GLST; b) penicillin, streptomycin, lincomycin, spectinomycin and minocycline (PSLSM) in whole milk used to dilute the contaminated raw semen followed by further dilution with glycerolated milk containing PSLSM; and c) penicillin, streptomycin, lincomycin and spectinomycin (PSLS) used with egg yolk Tris diluent in the same way as PSLSM and milk. Diluted semen was incubated at 35 degrees C for 5 or 40 min before cooling commenced. To assess the efficacy of the antibiotics in controlling P. aeruginosa, diluted semen samples were cultured for the organism before and after freezing. The GLST antibiotics added to raw semen and milk reduced the counts of P. aeruginosa before or after freezing. When egg yolk Tris was used, GLST inhibited the organism as indicated by its low growth in culture before freezing and absence of growth from samples after freezing. With PSLSM and PSLS treatments, the organism was recovered in milk and egg yolk Tris processed semen both before and after freezing. However, incubation at 35 degrees C for 40 min prior to cooling, compared to incubation of 5 min, appeared to reduce the bacterial counts after freezing.     

Evaluation of a monoclonal antibody to the K99 fimbrial adhesin produced by Escherichia coli enterotoxigenic for calves, lambs and piglets

All the K99+ Escherichia coli grown at 37 degrees C stained strongly with a peroxidase labelled K99 monoclonal antibody using a direct immunoperoxidase staining procedure. There was no reaction when these bacteria were cultured at 18 degrees C or when K99- E coli were grown at either temperature. The binding of the monoclonal antibody to K99 antigen was inhibited by OK antisera to heterologous K99+ E coli but OK antisera to E coli producing adhesins other than K99 were without effect. Using the slide agglutination test the reactions of the monoclonal antibody were identical to those of a polyclonal antiserum to K99 when both were used in parallel to examine 100 K99+ E coli from at least 10 somatic O groups and 1308 K99+ E coli from at least 82 different somatic O groups submitted for routine serological typing in England or the, USA. The monoclonal antibody reacted with K99+ E coli in cryostat sections of the ileum from a piglet infected with E coli strain B44 (O9: K30, K99, F41) but there was no reaction with similar material from piglets infected by E coli strains 1751 (O101: F41), X177/81 (O9: K103, 987P) or Abbotstown (O149: K91, K88ac).     

An attempt to induce mastitis in cows using Klebsiella pneumoniae isolated in the cow barn

The importance was studied of cowshed strains of K. pneumoniae as causative agents of bovine mastitis. An experiment was conducted with a healthy lactating dairy cow at the age of five years. The microbial inoculum was introduced by a teat tubule, at a dose of 2 ml broth culture 24 hours old. At first, Bac. subtilis was instilled in one udder quarter, to find out the local reaction to nutrient medium, and/or to an uninjurious saprophyte . After five days, a trial was performed on the other udder quarters using three strains of K. pneumoniae, isolated from the rectal contents and the skin of healthy cattle. Finally reinfection followed to study potential allergization of mammary gland. The conclusions as follows may be drawn from the results: 1) Bac. subtilis did not cause any alteration of the main clinic signs, but the inoculated quarter became swollen and its watery secretion contained pus corpuscles. No elimination of bacilli was observed and the morbid condition recovered in five to seven days. 2) After instillation of K. pneumoniae cultures bodily temperature rose by 1.9 degrees C in the course of six hours, all three udder quarters became swollen, showed symptoms of hyperthermia and pain , their secretion also exhibited pathological changes. The NK test revealed highly increased gelling of secretion and pH shift to alkalinity, the number of somatic cells increased 12 to 17 times. The main clinic signs, as well as appearance and consistence of the udder became normal in three days, but the other parameters like NK test and the number of somatic cells in milk normalized in about ten days. 3) Klebsiellae were eliminated from the quarters for one to eleven days, the elimination lasted longer and the counts were significantly higher in capsular strains.(ABSTRACT TRUNCATED AT 250 WORDS)     

Testing Milkofix, a new preservative preparation for milk samples used for infrared analysis of milk components. II. Verification of its preservative effects in relation to infrared analysis]

For the purposes of the infrared analysis of the basic milk composition, Milkofix, an ecologically friendly preparation used for milk sample preservation (Trzicky, 1990), was compared with untreated samples (N) and with samples preserved with sodium azide (A), bronopol (B) and potassium dichromate (C) at a storage temperature of 20 degrees C (I) and 4 degrees C (II) in samples kept for 14 and 18 days. Pursuant to the recommendations cited in literature, the preservatives had these concentrations: 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; M = 0.1250 g, all amounts are per 25 ml milk. Three bulk milk samples were used which were analyzed on an automatic Milko-Scan 133 B infraanalyzer (Foss Electric, Denmark) every day. On the basis of a graphical evaluation of the results by IDF recommendations (1985) the times within which the applicable results could be obtained were determined for the various methods of milk sample treatment (Figs. 1 to 6): N I--0 days; A I--9; B I--10; C I--13; M I--4; N II--10; A II--5; B II--11; C II--15; M II--10 (Tab. I). The results recorded for Milkofix are in agreement with the conclusions drawn in the previous study, where the intervals of four and nine days were determined. The days to milk sample coagulation were as follows: N I--1 day, M I--10 days. The coagulation in A I, B I, C I and N II samples was not observed even after 13-day storage and in A II, B II, C II and M II samples not even after 17 days of storage. The results for particular components (fat, proteins, lactose) of milk samples differently treated in time are presented in Tabs. II, III and IV. A system of evaluating criteria (Tab. V) was used to determine the order beginning from the most convenient method of milk sample treatment for the given purpose: 1. C II, 2. C I, 3. B II and A I, 4. B I, 5. M II, 6. N II, 7. A II, 8. M I and 9. N I.(ABSTRACT TRUNCATED AT 400 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 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.     

Survival of Yersinia enterocolitica in soil and water.

Yersinia enterocolitica O3, O4, O5A, O5B, O6, O6, 30, O9 and O13 and Yersinia intermedia were examined to clarify their survival in natural soil, river water and well water. The O3 strain disappeared most rapidly from soil at both temperature of 4 degrees C and 20 degrees C and from river water at 20 degrees C. Although the O5B and O9 strains disappeared before the O3 strain did from river water at 4 degrees C, the O9 strain survived as long as non-virulent strains did in soil at 4 degrees C. The O3, O5B and O9 strains survived longer at 4 degrees C than at 20 degrees C in soil and river water. Non-virulent strains of O4, O5A and O6 survived longer in well water than in soil and river water. Although the O3 and O5A strains disappeared from supernatant filtered with 100 microns and 5 microns pore size filters, they maintained their viable cell numbers in supernatant of soil filtered with 0.22 microns pore size filter and in the autoclaved supernatant.     

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.     

Bacterial growth during the early phase of infection determines the severity of experimental Escherichia coli mastitis in dairy cows

The aim of this study was to investigate the importance of bacterial growth for the severity of experimental Escherichia coli mastitis, indirectly expressed as the area under the curve of bacterial counts in milk over time. The association of pre-infusion somatic cell count and post-infusion influx of inflammatory cells in milk with severity of infection was also examined. Bacterial growth was studied through culture in milk samples (in vitro) and through monitoring of bacterial counts in milk during the early phase of infection (in vivo) in 36 cows. Individual variation in bacterial counts was more than 2 x 10(2)-fold after 6 h of in vitro incubation, and more than 8 x 10(2)-fold 6 h after intramammary infusion. In vitro growth in milk was not associated with in vivo growth during the early phase of infection, nor with severity of E. coli mastitis. Somatic cell count before experimental E. coli mastitis was negatively associated with in vivo bacterial growth during the early phase of infection (R2 = 0.28), but was not associated with severity of E. coli mastitis (R2 = 0.06). In vivo bacterial growth during the early phase of infection (positive association; R2 = 0.41), together with influx of inflammatory cells in milk, expressed as mean hourly increase of somatic cell count between 6 and 12 h post-infusion (negative association; R2 = 0.11), are major determinants for the severity of experimental E. coli mastitis (R2 = 0.56).     

Effect of brewer's grain on rumen fermentation, milk production and milk composition in lactating dairy cows

Six lactating Holstein cows were divided into two groups (n = 3) and used in a double reversal trial with three periods of 14 days each to evaluate the rumen fermentation, milk production and milk composition of cows fed brewer's grain (BG). The control diets contained 14% chopped Sudangrass hay, 24% corn silage, 18% alfalfa hay cube, 34% concentrate mixture‐1 and 10% concentrate mixture‐2 (wheat bran, soybean meal and cottonseed). In the experimental diet, wet BG replaced the concentrate mixture‐2. The protozoal population, concentration of ammonia‐N and volatile fatty acids in the ruminal fluid did not differ between the control and BG diets. The molar percentage of acetic acid was significantly higher (P < 0.05) with the BG diet at 5 h after feeding. The milk yield, the percentage of protein, lactose, solids not‐fat and somatic cell counts of milk did not differ between the two diets. The percentage of milk fat tended to increase with the BG diet. The BG diet significantly increased the proportions of C18:0 and C18:1 in milk fat (P < 0.01, P < 0.05, respectively) and tended to increase that of conjugated linoleic acid.     

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.     

A clinical trial to evaluate the effectiveness of antibiotic treatment of lactating cows with high somatic cell counts in their milk

OBJECTIVE: To determine the effectiveness of treatment of lactating cows with high somatic cell counts in milk. DESIGN: Randomised clinical trial. METHODS: Single pooled quarter samples of milk were obtained from cows with somatic cell counts above 500,000 cells/mL on fifty farms. Milk samples were cultured for known mastitis bacterial pathogens. Cows were randomly allocated to treated and untreated groups. Treated cows received both intramammary cloxacillin and parenteral erythromycin. Single pooled quarter milk samples were obtained at 6 weeks after treatment and were cultured for the presence of pathogenic bacteria. The percentage of samples with no growth at the post-treatment culture was used as an estimate of the bacteriological cures for each pathogen type and for each treatment group. Somatic cell counts of cows were compared between treatment groups and within pathogen group. The number of cows that completed a full lactation were compared between each treatment group and within each pathogen group. RESULTS: Treatment had no effect upon bacteriological cures, irrespective of pathogen present or the presence of bacteria during the previous lactation. There was no effect of treatment upon somatic cell count except for cows infected with Streptococcus dysgalactiae in which treatment caused a significant lowering of cell counts. This effect was not present in the subsequent lactation. Treatment of chronically infected cows did not alter the probability of a cow completing a full lactation but did improve the probability of newly infected cows being retained for the next lactation. Twenty-eight of 214 treated cows developed clinical mastitis in more than one quarter after treatment, thus indicating a poor technique by farmers for the insertion of intramammary antibiotics. CONCLUSIONS: Treatment during lactation of cows with high somatic cell counts in milk is ineffective in reducing bacterial infections and in reducing somatic cell counts to acceptable numbers.