Direct Microscopic Somatic Cell Count Research Articles

Comparison of California mastitis test and Draminski mastitis detector as on-farm methods for monitoring udder health in lactating buffalo

This study was conducted to compare the performance of the California Mastitis Test (CMT) and Draminski Mastitis Detector (DMD) for monitoring udder health in lactating buffalo. A total of 647 milk samples collected from 145 apparently healthy lactating buffalo cows during the first three months of the lactation period were used in this study. Both DMD and CMT were performed on-farm directly after sampling of the milk. The diagnostic accuracy of CMT and DMD was evaluated by estimating the area under the Receiver-Operating Characteristic curve (AUC) based on a value of 200,000 somatic cells per mL as a cut-point value for an infected quarter detected by direct microscopic somatic cell count. Data analysis showed that 63.76% of CMT results were “Trace” and 99% of DMD readings were greater than 300. The accuracy of CMT and DMD was close to each other; the AUC was 80.1% and 81.4% for CMT and DMD, respectively. The optimal cut-point value for CMT was Trace (T), and 515 for DMD. In conclusion, both CMT and DMD are practical on-farm tests for identifying infected udder in buffalo cows, and the suggested cut-point value for DMD is 515 for buffalo’s milk testing instead of the 300 that was assigned by the manufacturer for cow’s milk.

The Effect of TEAT PROTECT spray and potassium permanganate teat dip in curing subclinical mastitis in crossbred cows of Villupuram district of Tamilnadu

A study was undertaken to assess the incidence of subclinical mastitis in crossbred cows of Villupuram district by using direct microscopic Somatic cell count (SCC). For this purpose sixty eight crossbred cows positive for subclinical mastitis (SCM) as per IDF (International Dairy Federation) criteria were randomly allotted in to two treatment groups. The cows in T1 group were subjected to post milking teat dip with one per cent Potassium permanganate (KMnO4) solution immediately after milking. The cows in T2 group were sprayed with TEAT PROTECT sprays. Sixteen healthy cows were kept as control group and udder was washed with plain water. The treatments were carried out for 30 days. Udder health status was determined by recording the parameters viz. somatic cell count (SCC), average daily milk yield, udder colony count and teat colony count. The results revealed that TEAT PROTECT spray is superior to Potassium permanganate in curing SCM of crossbred cows.

Short communication: Evaluation of confirmatory stains used for direct microscopic somatic cell counting of sheep milk

Current FDA regulatory screening and confirmatory methods, electronic cell counting and the direct microscopic somatic cell count (DMSCC), differ for the detection of abnormal milk in sheep and goats. The DNA-specific electronic SCC screening methods such as Fossomatic (Foss, Hillerød, Denmark) can be used for both sheep and goat milk; however, the nonspecific methylene blue-based stains used for DMSCC in sheep cannot be used for goats as they nonspecifically stain cytoplasmic particles naturally present in goat milk. The DNA-specific stain pyronin Y-methyl green (PMG) is currently used for DMSCC in goats. Sheep also shed cytoplasmic particles during the milk secretory process, but in fewer numbers than goats. The objective of this study was to determine whether the nonspecific, methylene blue-based Levowitz-Weber (L-W) stain is the appropriate regulatory stain to use for DMSCC in sheep milk. Composite milk samples from 42 commercial dairy ewes were collected every 4 wk for the duration of each ewe's lactation for a total of 10 sample days. Milk samples were subjected to 3 methods of SCC determination: automated Fossomatic counting, DMSCC with L-W stain, and DMSCC with PMG stain conducted according to FDA regulatory procedures (2400 series forms). The DMSCC from milk smears stained with L-W were greater than those from smears stained with PMG and those from the Fossomatic analysis on 6 of the 10 sampling days. Milk smears stained with PMG did not differ from Fossomatic analysis at any sampling. The average milk SCC for L-W, PMG, and Fossomatic were (mean ± SE) 275±36 × 103, 174±24 × 103, and 164±24 × 103 cells/mL, respectively. The DMSCC for milk stained with L-W was 58% greater than that with PMG and 68% greater than that obtained with the Fossomatic analysis. In conclusion, DMSCC of sheep milk stained with the nonspecific, methylene blue L-W stain resulted in a marked increase in SCC over that of the DNA-specific stain PMG and Fossomatic SCC analysis. The findings of this study support the continued use of Fossomatic SCC but recommend the replacement of the methylene blue-based stains with DNA-specific PMG for determination of DMSCC in sheep milk.

CONTAGEM AUTOMÁTICA E MICROSCÓPICA DIRETA DAS CÉLULAS SOMÁTICAS DO LEITE DE OVELHAS DA RAÇA LACAUNE, UTILIZANDO COMO CORANTES O ROSENFELD E VERDE DE METIL E PIRONINA-Y

The objectives of the present study were the analysis of cell counts, the comparison between different methods for counting cells and the quantification of different types of cells observed in the milk of Lacaune sheep, using automatic somatic cell counts (SCC) and direct microscopic SCC. Milk smears were stained with methyl green-pyronin-Y and Rosenfeld stain. Mean of automatic cell counts was 0.145 x106 cells/mL of milk, and mean of direct microscopic counts using methyl green-pyronin-Y and Rosenfeld were respectively 0.157 and 0.193 x106 cells /mL of milk. Samples used in direct microscopic counts were evaluated according to the different types of cells and the following results were recorded: 0.094 and 0.14 x106 mononuclear cells /mL, 0.063 and 0.051 x106 polymorphonuclear cells /mL of milk using methyl green-pyronin-Y and Rosenfeld stain, respectively. It may be concluded that there were differences in the methods used for counting cells. The number of cells recorded was lower when methyl green-pyronin-Y and automatic counts were used, compared with direct microscopy using Rosenfeld stain. KEY WORDS: somatic cells and cytology, lacaune, milk, sheep.

Relationships Between Somatic Cell Count and Udder Traits in Jersey Cows

Kul, E. and Erdem, H. 2008. Relationships between somatic cell count and udder traits in Jersey cows. J. Appl. Anim. Res, 34: 101–104. To determine the relationships between some udder traits and somatic cell count (SCC), 107 Jersey cows raised at Karakoy State Farm, Samsun, Turkey, were used. Milk samples and udder measures were taken from cows at 70±14, 140±14 and 210±14 days in milk. SCC was determined by direct microscopic somatic cell counting (DMSCC). SCC values increased with advancing lactation. Significant correlations (P<0.05, P<0.01) were estimated between udder traits, udder scores with SCC, overall or at different lactation stages. Thus, it was concluded that front udder attachment and udder depth can be effectively used for reducing SCC.

Bovine Mastitis in Zebu and Crossbred Cattle under the Extensive Management System in Tanzania

A study was carried out to evaluate the incidences and causes of bovine mastitis in Tanzanian shorthorn zebu (Bos indicus) in the traditional sector and crossbred cows (Bos taurus×Bos indicus) in the dairy ranching sector, both found under the extensive range management system. Management practices were evaluated through a survey study using structured questionnaires. A total of 120 lactating cows (60 cows from each sector) were screened for the disease using the California Mastitis Test (CMT). Confirmatory tests used for infected cows included; the Direct Microscopic Somatic Cell Count (DMSCC), culture, bacteriological and biochemical laboratory assays. Survey results showed that management practices were generally very poor in both sectors with 84% of the surveyed herds being kept and milked under very unhygienic environmental conditions. The level of infection was higher in the crossbred cows (5% clinical and 38.3% sub-clinical mastitis) and lower in the zebu cows with only sub-clinical mastitis (23.3%). Crossbred cows had (p<0.05) higher somatic cell counts than zebu cows. The four highest-ranking bacterial isolates in order of importance were Staphylococcus aureus, Escherichia coli, Streptococcus agalactiae and Bacillus spp. It was concluded that bovine mastitis under the extensive management system in Tanzania was a result of poor management practices and that zebu cows were more resistant to the diseases than crossbred cows. (Asian-Aust. J. Anim. Sci. 2002. Vol 15, No. 5 : 751-756)

Subclinical mastitis in ewes and its effect on lamb performance.

Two studies were conducted to 1) assess the effectiveness of the California mastitis test (CMT) relative to direct microscopic somatic cell count (DMSCC) and(or) somatic cell count (SCC) procedures for detecting subclinical mastitis in ewes, 2) determine the incidence of subclinical mastitis based on repeated or single sample measures and organisms associated with the inflammation, and 3) assess the relationship between milk quality measures and lamb performance. The relationship between DMSCC and SCC scores was significant (P less than .01); 90% of the variation in DMSCC scores was accounted for by SCC scores. In contrast, CMT scores accounted for only 26% of the variation in DMSCC and 30 to 34% of the variation in SCC scores. Incidence of inflammation varied from 17 to 50% of ewes tested, depending on the study and the method of assessment. Staphylococcus species were cultured from 14/41 samples tested, with cultures of Streptococcus species (3/41) and Micrococcus species (1/41) also present. The effect of subclinical mastitis in ewes on lamb performance was minimal when assessed by regressing lamb weights on subclinical mastitis and milk quality scores. In conclusion, growth performance of lambs in a management system where they had access to supplemental feed was not influenced by the quality of milk produced by ewes, or by the degree of subclinical mastitic inflammation present when they suckled.

Warm vs Cold Sampling of Raw Milk for Direct Microscopic Somatic Cell Counting

Standard Methods for the Examination of Dairy Products (1) requires that milk be sampled cold for analysis of both bacteria and somatic cells by the direct microscopic method. Preliminary observations made at Dairy Quality Control Institute, Inc. indicated that better precision in somatic cell counting could be obtained if milk was sampled at 38°C rather than 0–4.4°C. A study was undertaken to confirm preliminary findings. Three technicians made direct microscopic somatic cell counts (DMSCC) in triplicate on milk samples standardized to four different levels of counts. The grand average counts at the four levels of cells for cold vs warm milk sampling were, in thousands of cells per ml, 149 vs 154, 410 vs 416, 705 vs 697, and 1034 vs 1024. Grand average standard deviations for cold vs warm, respectively, at the four levels of count were 19 vs 17, 47 vs 42, 54 vs 50, and 69 vs 62. Thus, an improvement in precision of counting was observed at all four levels of somatic cells when raw milk is warmed to 38°C prior to sampling.

Collaborative Study to Determine Effects of Milk Composition on Somatic Cell Counts

Quarter, composite, and bulk tank milk samples were analyzed in a three laboratory collaborative study to determine the relationship of milk fat and protein content with milk somatic cell counts. Milk somatic cell counts were determined by two Coulter counters, a Fossomatic counter, and by direct microscopic somatic cell counting. In general, variability among somatic cell counts measured by different procedures was not related to protein or fat content of milk. The greatest percentage of variation between counts that could be explained by fat content of milk was 20.2% between a Coulter and direct microscopic somatic cell counts. The greatest percentage of variation between counts that could be explained by protein content of samples was 12.9% between a Coulter and Fossomatic counts. Breed of cow from which samples were collected also had little influence on differences among milk somatic cell counts. Differences among milk somatic cell counts due to counting methods did vary among quarter, composite, and bulk tank milk samples.

Variability in True Protein, Casein, Nonprotein Nitrogen, and Proteolysis in High and Low Somatic Cell Milks

Monthly variation in milk protein (total nitrogen×6.38) and milk fat was determined for 115 farms over 2 yr. Yearly average milk protein and fat tests in each year were 3.16 and 3.62%, respectively. The mean regression coefficient for milk protein with respect to milk fat was .47 for the entire period.Twenty-four farms were selected and grouped high or low based on their previous 2-yr somatic cell history. Monthly milk samples for each farm were tested for direct microscopic somatic cell count, total nitrogen, noncasein nitrogen, and nonprotein nitrogen. No differences in monthly nonprotein nitrogen, true protein, and casein were found between groups. Casein as a percent of total nitrogen was significantly higher for the low somatic group for seven of the 12 mo studied but was significantly higher for 9 mo when expressed as a percent of true protein. The average increase in tyrosine value for incubated preserved milk was significantly higher for the high somatic cell milk, indicating higher proteolytic activity in high somatic cell milk. Electrophoretic analysis of high and low somatic cell milk indicated that there was substantial proteolytic breakdown of αS-casein and β-casein by proteases associated with elevated somatic cell counts.

Preparation and Use of Somatic Cell Count Samples (SCCS) for Comparison of Milk Somatic Cell Counting Methods

Somatic cell count samples (SCCS) for use in comparison of milk somatic cell counting methods were prepared from the cell sediment deposited in a creamery milk separator. Bovine milk somatic cells were resuspended from the sediment, and serial cell dilutions were prepared in bronopol-preserved milk diluent. Over a 1-year period, sets of SCCS were prepared each month and sent to milk-testing laboratories in the U.S.A., Canada and Europe, and counted by the methods in use at those Laboratories: (a) direct microscopic somatic cell count (DMSCC), (b) Fossomatic counter and (c) Coulter counter. Cell counts were normalized to eliminate the effect of month to month variation in the cell content of the SCCS. Counts obtained by the three methods were similar, although Coulter counter results tended to be lower, and significantly lower (P< 0.05) in SCCS with cell counts greater than 700,000 cells/ml than those counts by the other two methods. The effect of shipping on SCCS stability was assessed for SCCS samples sent to and returned from other laboratories, and counted by the Fossomatic method on their return. Counts were similar before and after shipping, except that results for SCCS with cell counts greater than 1,000,000 cells/ml were significantly higher (P<0.05) after their return.

Somatic Cell Reference Samples for Calibration of Milk Somatic Cell Counting Methods

Somatic cell count samples (SCCS) for use in calibration of milk somatic cell counting methods were prepared from raw bulk milk preserved with potassium dichromate. Somatic cells were separated by centrifugation, then appropriate cell dilutions were prepared in the dichromate-preserved skim milk. Somatic cell counts from SCCS stored at 4°C were stable over a 23-wk period. No bacterial contamination was detected in these samples. In a collaborative study among eight laboratories, SCCS were not affected by usual conditions by shipping. The SCCS can be used as reference standards for the direct microscopic somatic cell count and the Fossomatic and Coulter Counter somatic cell counting methods.

Comparison of Semiautomated Method with Official Optical Somatic Cell Counting Method III for Determining Somatic Cells in Milk

The new method specifying the Fossomatic-90 differs from the official method, 46.105-46.109, in that the modified instrument includes a halogen lamp; a semiconductor photoelectric detector; a less expensive, bench-top cabinet; manual injection of a larger sample, and a reduced capacity. The new instrument was compared with 2 optical somatic cell counters in routine use. On each of 3 days, 12 subsamples were prepared for each of 5 cell count levels from AM milk with half kept fresh and half preserved with 0.05% potassium dichromate. Subsamples were refrigerated and read 30+ h post-collection. Duplicate sets were read in random order on each machine daily (CV 0.77%). Two sets of slides read by 2 technicians each (strip reticle on 2 smears/slide) gave geometric mean direct microscopic somatic cell count (DMSCC) levels of 296, 526, 772, 930, and 1438 th/mL. Within-technician CV values (from day-level means) ranged from 1.68 to 2.28%. Geometric mean cells in th/mL on the new machine were significantly higher than those on the other two (674 vs 621) and were closer to the DMSCC (694). On the new machine, cell counts were 8.5% greater than on the original machines, were only 2.9% lower than the DMSCC, and showed no significant evidence of bias. Preserved samples averaged slightly greater than fresh (5.3%) but only on the original machines. Carryover by covariance analysis was insignificant. Except for cell levels, high machine precision (error CV value of 1.18%) gave differences with statistical but not practical significance, even for regulatory laboratories.

Preparation, Use and Evaluation of Standards Developed for Simultaneous Monitoring of Coulter and Fossomatic Electronic Cell Counting Instruments in Ontario

Special standards for Somatic Cell Counting (SCC) were developed at the Central Milk Testing Laboratory in Guelph for Ontario laboratories performing the tests. One laboratory used a Coulter milk counter for composite herd samples, two laboratories used five Fossomatic counters for cow samples, while the fourth used a Coulter counter for research purposes. Standards were prepared at low, medium and high count levels from fresh herd milks containing somatic cells near levels of 100,000, 500,000 and 1,000,000/ml. As the lifespan of the suspensions of somatic cells was three months, they were prepared in sufficient quantities to test daily for 3 months, and to be used throughout the day for calibration maintenance. The actual counts in the standards were established by direct microscopic somatic cell count (DMSCC) performed in two laboratories simultaneously with exchange of slides. Counts were re-confirmed every week by the same DMSCC procedures. The standards are suitable for initial calibration, daily calibration maintenance, daily instrument performance appraisal and trouble-shooting. In daily use each laboratory tests each standard five times each morning on each instrument. The same vials are used throughout the day, every 0.5 h for appraisal of continuous calibration and instrument function. The mean and standard deviations of standards are determined and compiled for each day.

Effect of Parity, Stage of Lactation, and Intramammary Infection on Concentration of Somatic Cells and Cytoplasmic Particles in Goat Milk

Aseptic foremilk samples (about 15ml) were collected biweekly for four samplings and then monthly throughout lactation from 35 does in three herds on Dairy Herd Improvement test. Bacteriology, direct microscopic somatic cell counts, and cytospin differential cell counts were performed on all samples. Milk yield was obtained from monthly Dairy Herd Improvement records. Thirty-nine percent of udder halves were infected, and coagulase negative staphylococci were the most frequent isolates (201 of 205). Somatic cell counts were influenced by lactation number, stage of lactation, and intramammary infection. Also, infection of one udder half increased somatic cells in the corresponding uninfected mammary half of the same animal. Cytoplasmic particles were not affected by stage of lactation or by intramammary infection but were greater for goats in first lactation. Milk yield was influenced by lactation number, stage of lactation, and intramammary infection.

Screening tests for abnormal milk and physical examination of the udder

Many tests have been described for use in detecting abnormal milk. These include: black plate or strip cup, catalase test, (modified) Whiteside test, serum albumin, milk chloride, electrical conductivity, California Mastitis Test (CMT), Wisconsin Mastitis Test (WMT), Direct Microscopic Somatic Cell Count (DMSCC) and Electronic Somatic Cell Counts (ESCC). In summary, the black plate is still a useful tool for the examination of lactating cows to detect clinical mastitis.&#x0D; The CMT Test is a useful cowside test for detecting subclinical infections.&#x0D; ESCC provides useful information for assessing the prevalence of subclinical infection and for regular evaluation of mastitis control practices.&#x0D; Examination of the udder may be indicated. Udder palpation is not as important in the current scheme of management of cows as it may have been in the past but could still provide useful additional indications for culling decisions.&#x0D; Teat end lesions may be reduced by avoiding chemical irritation and faulty machine function. However, teat end appearance may be related to high production and, depending on degree, is not always an indication of unusual trauma.

Reliability and Analyst Performance Limits for the Field and Single Strip Direct Microscopic Somatic Cell Count Procedure

The means and total components of variance were compared for the field and single strip direct microscopic somatic cell count (DMSCC) procedures. The field count procedure averaged 12 – 28% higher than the single strip count procedure in the 300,000 to 1,200,000 DMSCC/ml range. The sum of the components of variance in logarithm units for the field procedure was 0.01058 (1485 degrees of freedom) with a coefficient of variation of 24%, whereas the sum for the single strip procedure was 0.00834 (2834 degrees of freedom) with a coefficient of variation of 21%. This study demonstrates that the single strip procedure yields more reliable and less variable results than does the field procedure.

Differentiation and Enumeration of Somatic Cells in Goat Milk

Non-leukocytic cell-like particles commonly observed in goat milk were examined ultrastructurally and cytochemically. Transmission electron microscopy indicated that these particles were generally membrane-bound and anucleate. They contained granular material in the dilated cisternae of the endoplasmic reticulum and homogeneous electron translucent inclusions that resembled lipid. Histochemical and fluorescent staining indicated that the particles contained large amounts of protein, some lipid, but no deoxyribonucleic acid. Several methods routinely used for estimating somatic cell counts in cow milk were compared to determine which one would give accurate estimates of somatic cell counts in goat milk. No significant difference was found (P> .05) among methods which specifically measure deoxyribonucleic acid. These included Membrane Filter-DNA, direct microscopic somatic cell counts using Pyron in Y-methyl green stain, and Fossomatic cell counts. Results of the Wisconsin Mastitis Test did not differ significantly from Fossomatic cell counts. Because Coulter electronic counts and direct microscopic somatic cell counts using Levowitz-Weber stain could not differentiate between the cell-like particles and the actual leukocytes, these methods resulted in elevated cell counts that were highly variable. Results indicate that only those counting methods that are specific for deoxyribonucleic acid can distinguish cell-like particles from somatic cells, and thereby give reliable estimates of somatic cell numbers in goat milk.

Milking Vacuum Influencing Indicators of Udder Health1,2

In 23 first-calf heifers we ascertained effects that milking vacuums of 34, 42.5, and 51 kPa had on udder health by percentage of quarter-samples positive for Staphylococcus aureus, total counts of bacteria on blood agar, teat end scores, California Mastitis Test, and direct microscopic somatic cell count.Measures were higher for 51 kPa than for 34 or 42.5 kPA. Samples from cows milked at 42.5 kPa had a lower percentage of samples positive for Staphylococcus aureus, lower total count, lower California Mastitis Test, and lower direct microscopic somatic cell count than samples from cows milked with 34 kPa. Teat end scores increased with increase in milking vacuum. Approximately 49% of the samples from cows milked with 51 kPa were positive for Staphylococcus aureus compared with 19.9 and 16.7% for 34 and 42.5 kPa. Milking vacuum of 51 kPa resulted in the highest percentage of cows with three (44.5%) and four (24.1%) quarters positive for Staphylococcus aureus. A milking vacuum of 51 kPa could increase mastitis in a herd having Staphylococcus aureus as the prevalent microorganism causing mastitis.

Variation in Food Microbiological Tests Used to Evaluate Analyst Performance

Procedures for evaluating analyst performance in state and federal milk and food laboratory quality assurance programs depend on the estimates of variation for the methods used. The total components of variance for a method consist of replicate error, among-analysts variance, and analyst-sample interaction components. Eleven methods were evaluated in this study. Total variance estimates of milk analytical procedures for the Standard Plate Count, plate loop count, coliform plate count, direct microscopic somatic cell count, electronic somatic cell count and Wisconsin mastitis test were 0.01045, 0.01371, 0.01590, 0.01265, 0.00504 and 4.06, respectively. Estimates of the logarithmic total variance of food analytical procedures for the aerobic plate count, coliform most probable number (MPN), Escherichia coli MPN, fecal coliform MPN and Staphylococcus aureus MPN were 0.00853, 0.10205, 0.14705, 0.14780 and 0.24245, respectively. The study showed that current analyst performance levels used by state and federal laboratory quality assurance programs are satisfactory.